Polypeptides having transgalactosylating activity

ABSTRACT

The present invention relates to polypeptides, specifically polypeptides having transgalactosylating activity and nucleic acids encoding these, and their uses in e.g. dairy product.

FIELD OF THE INVENTION

The present invention relates to polypeptides, specifically polypeptideshaving transgalactosylating activity and nucleic acids encoding these,and their uses in e.g. dairy product.

BACKGROUND OF THE INVENTION

Galactooligosaccharides (GOS) are carbohydrates which are nondigestablein humans and animals comprising two or more galactose molecules,typically up to nine, linked by glycosidic bonds. GOS's may also includeone or more glucose molecules. One of the beneficial effects of GOS's istheir ability of acting as prebiotic compounds by selectivelystimulating the proliferation of beneficial colonic microorganisms suchas bacteria to give physiological benefits to the consumer. Theestablished health effects have resulted in a growing interest in GOSsas food ingredients for various types of food.

The enzyme β-galactosidase (EC 3.2.1.23) usually hydrolyses lactose tothe monosaccharides D-glucose and D-galactose. In the normal enzymereaction of β-galactosidases, the enzyme hydrolyses lactose andtransiently binds the galactose monosaccharide in a galactose-enzymecomplex that transfers galactose to the hydroxyl group of water,resulting in the liberation of D-galactose and D-glucose. However, athigh lactose concentrations some β-galactosidases are able to transfergalactose to the hydroxyl groups of D-galactose or D-glucose in aprocess called transgalactosylation whereby galacto-oligosaccharides areproduced. Also at high lactose concentrations some β-galactosidases areable to transfer galactose to the hydroxyl groups of lactose or higherorder oligosaccharides.

The genus Bifidobacterium is one of the most commonly used types ofbacteria cultures in the dairy industry for fermenting a variety ofdiary products. Ingestion of Bifidobacterium-containing productsfurthermore has a health-promoting effect. This effect is not onlyachieved by a lowered pH of the intestinal contents but also by theability of Bifidobacterium to repopulate the intestinal flora inindividuals who have had their intestinal flora disturbed by for exampleintake of antibiotics. Bifidobacterium furthermore has the potential ofoutcompeting potential harmful intestinal micro-organisms.

Galacto-oligosaccharides are known to enhance the growth ofBifidobacterium. This effect is likely achieved through the uniqueability of Bifidobacterium to exploit galacto-oligosaccharides as acarbon source. Dietary supplement of galacto-oligosaccharides isfurthermore thought to have a number of long-term disease protectingeffects. For example, galacto-oligosaccharide intake has been shown tobe highly protective against development of colorectal cancer in rats.There is therefore a great interest in developing cheap and efficientmethods for producing galacto-oligosaccharides for use in the industryfor improving dietary supplements and dairy products.

An extracellular lactase from Bifidobacterium bifidum DSM20215 truncatedwith approximately 580 amino acids (BIF3-d3) has been described as atransgalactosylating enzyme in a solution containing lactose solubilisedin water (Jorgensen et al. (2001), Appl. Microbiol. Biotechnol., 57:647-652). WO 01/90317 also describes a truncation variant (OLGA347) asbeing a transgalactosylating enzyme and in WO 2012/010597 OLGA347 wasshown to transfer a galactose moiety to D-fucose, N-acetyl-galactosamineand xylose.

In WO 2009/071539 a differently truncated fragment compared to BIF3-d3is described as resulting in efficient hydrolysis and very lowproduction of GOS when tested in milk.

The Bifidobacterium bifidum lactase enzymes described above have thedrawback of either requiring high lactose concentrations such as above10% (w/w) in order to produce GOS, or a high surplus of another acceptormolecule to generate heterooligosaccharides. Furthermore, a molecule hasbeen described that predominately having beta-galactosylase (hydrolase)activity.

There is still a need to develop enzymes that are efficient at producingGOS in applications with low lactose substrate levels such as in milk.

SUMMARY OF THE INVENTION

It is an object of embodiments of the invention to provide a polypeptidewhich has a useful ratio of transgalactosylation to hydrolysis activityand thus are efficient producers of GOS when incubated with lactose evenat low lactose levels such as in a milk-based product. It is a furtherobject of embodiments of the invention to provide a method forproduction of galacto-oligosaccharides (GOS) in situ in dairy products.It is a further object of embodiments of the invention to provide amethod for developing a cheaper and more efficient method for productionof galacto-oligosaccharides (GOS) for use in the industry. It is furtherobject of embodiments of the invention to provide polypeptides which arestable against further truncation such as by proteolytic degradationwhen produced in a suitable organism such as Bacillus subtilis e.g.Bacillus subtilis strain BG3594. It is yet a further object ofembodiments of the invention to provide polypeptides which are stableagainst further truncation during storage after final formulation.

The present inventors have surprisingly found that the polypeptidesdisclosed herein are efficient producers of galacto-oligosaccharides forexample in situ when incubated in a lactose containing composition suchas milk, wherein they have an efficient conversion of lactose into GOSresulting in a lower amount of free lactose. The presence ofgalacto-oligosaccharides in diary products or other comestible productshas the advantage of enhancing the growth of beneficial microbialstrains (probiotics) such as the health-promoting Bifdobacterium sp. inthe product itself and/or in the human or animal that consumes theproduct.

In one aspect, disclosed herein is a polypeptide havingtransgalactosylating activity, which comprises an amino acid sequencehaving at least 90% sequence identity with SEQ ID NO: 1, and whereinsaid polypeptide, when being an expression product in a Bacillussubtilis strain BG3594 of a nucleic acid sequence, which encodes saidpolypeptide, is the only polypeptide expression product of said nucleicacid sequence that exhibits transgalactosylating activity.

In one aspect, disclosed herein is a polypeptide havingtransgalactosylating activity selected from the group consisting of:

-   -   a. a polypeptide comprising an amino acid sequence having at        least 90% sequence identity with SEQ ID NO: 1, wherein said        polypeptide consists of at most 980 amino acid residues,    -   b. a polypeptide comprising an amino acid sequence having at        least 97% sequence identity with SEQ ID NO: 2, wherein said        polypeptide consists of at most 975 amino acid residues,    -   c. a polypeptide comprising an amino acid sequence having at        least 96.5% sequence identity with SEQ ID NO: 3, wherein said        polypeptide consists of at most 1300 amino acid residues,    -   d. a polypeptide encoded by a polynucleotide that hybridizes        under at least low stringency conditions with i) the nucleic        acid sequence comprised in SEQ ID NO: 9, 10, 11, 12 or 13        encoding the polypeptide of SEQ ID NO: 1, 2, 3, 4, or 5; or ii)        the complementary strand of i),    -   e. a polypeptide encoded by a polynucleotide comprising a        nucleotide sequence having at least 70% identity to the        nucleotide sequence encoding for the polypeptide of SEQ ID NO:        1, 2, 3, 4 or 5 or the nucleotide sequence comprised in SEQ ID        NO: 9, 10, 11, 12 or 13 encoding a mature polypeptide, and    -   f. a polypeptide comprising a deletion, insertion and/or        conservative substitution of one or more amino acid residues of        SEQ ID NO: 1, 2, 3, 4 or 5.

In another aspect disclosed herein is a polypeptide havingtransgalactosylating activity selected from the group consisting of:

-   -   a. a polypeptide comprising an amino acid sequence having at        least 96.5% sequence identity with SEQ ID NO: 3, wherein said        polypeptide consists of at most 1300 amino acid residues,    -   b. a polypeptide comprising an amino acid sequence having at        least 90% sequence identity with SEQ ID NO: 1, wherein said        polypeptide consists of at most 980 amino acid residues,    -   c. a polypeptide encoded by a polynucleotide that hybridizes        under at least low stringency conditions with i) the nucleic        acid sequence comprised in SEQ ID NO: 9, 10, 11, 12 or 13        encoding the polypeptide of SEQ ID NO: 1, 2, 3, 4, or 5; or ii)        the complementary strand of i),    -   d. a polypeptide encoded by a polynucleotide comprising a        nucleotide sequence having at least 70% identity to the        nucleotide sequence encoding for the polypeptide of SEQ ID NO:        1, 2, 3, 4 or 5 or the nucleotide sequence comprised in SEQ ID        NO: 9, 10, 11, 12 or 13 encoding a mature polypeptide, and    -   e. a polypeptide comprising a deletion, insertion and/or        conservative substitution of one or more amino acid residues of        SEQ ID NO: 1, 2, 3, 4 or 5.

In one aspect, disclosed herein is polypeptide which is a C-terminaltruncated fragment of SEQ ID NO:22 having transgalactosylating activityand which are stable against further truncation such as by proteolyticdegradation when produced in a suitable organism such as Bacillussubtilis e.g. Bacillus subtilis strain BG3594 and/or which are stableagainst further truncation during storage after final formulation.

In one aspect, disclosed herein is a polypeptide comprising an aminoacid sequence having at least 90% sequence identity with SEQ ID NO: 1,wherein said polypeptide consists of at most 980 amino acid residues.

In one aspect, disclosed herein is a polypeptide comprising an aminoacid sequence having at least 97% sequence identity with SEQ ID NO: 2,wherein said polypeptide consists of at most 975 amino acid residues, isprovided.

In one aspect, disclosed herein is a polypeptide comprising an aminoacid sequence having at least 96.5% sequence identity with SEQ ID NO: 3,wherein said polypeptide consists of at most 1300 amino acid residues.

In one aspect, disclosed herein is a nucleic acid capable of encoding apolypeptide as described herein.

In one aspect, disclosed herein is an expression vector and/or a plasmidcomprising a nucleic as described herein, or capable of expressing apolypeptide as described herein.

In one aspect, disclosed herein is a cell capable of expressing apolypeptide as described herein.

In one aspect, disclosed herein is a method of expressing a polypeptide,the method comprising obtaining a cell as described herein andexpressing the polypeptide from the cell, and optionally purifying thepolypeptide.

In one aspect, disclosed herein is a composition comprising apolypeptide as described herein, preferably a food composition, morepreferably a dairy product.

In one aspect, disclosed herein is a method for producing a food productsuch as a dairy product by treating a milk-based substrate comprisinglactose with a polypeptide as described herein.

In one aspect, disclosed herein is a galacto-oligosaccharide orcomposition thereof obtained by treating a substrate comprising lactosewith a polypeptide as described herein.

LEGENDS TO THE FIGURE

FIG. 1 shows a plasmid map for the BIF_(—)1326 variant for recombinantexpression in Bacillus subtilis.

FIG. 2 shows SDS-PAGE showing truncation variants purified using HyperQcolumn eluted with a NaCl gradient.

FIG. 3 shows the ratio of transgalactosylation activity. Ratio iscalculated as ratio between Abs420 with acceptor present divided byAbs420 without acceptor present times 100. Variants at or below index100 are purely hydrolytic variants, whereas the level above reflectsrelative transgalactosylating activity.

FIG. 4 shows Galacto-oligosaccharides (GOS) generating efficacy ofselected variants in a yoghurt matrix at 30° C. for 3 hours. In thisexample GOS is the accumulative amount oligosaccharides at and aboveDP3.

FIG. 5 shows SDS-PAGE gel showing the different variants from table 2expressed and the degradation fragments detected. Lower panel showsmagnification and identification of degradation bands.

SEQUENCE LISTING

SEQ ID NO: 1 (also named (BIF_(—)917) herein) is a 887 amino acidtruncated fragment of SEQ ID NO: 22.

SEQ ID NO: 2 (also named (BIF_(—)995) herein) is a 965 amino acidtruncated fragment of SEQ ID NO: 22.

SEQ ID NO: 3 (also named (BIF_(—)1068) herein) is a 1038 amino acidtruncated fragment of SEQ ID NO: 22.

SEQ ID NO: 4 (also named (BIF_(—)1172) herein) is a 1142 amino acidtruncated fragment of SEQ ID NO: 22.

SEQ ID NO: 5 (also named (BIF_(—)1241) herein) is a 1211 amino acidtruncated fragment of SEQ ID NO: 22.

SEQ ID NO: 6 (also named (BIF_(—)1326) herein) is a 1296 amino acidtruncated fragment of SEQ ID NO: 22.

SEQ ID NO: 7 is Bifidobacterium bifidum glycoside hydrolase catalyticcore

SEQ ID NO: 8 is a nucleotide sequence encoding an extracellular lactasefrom Bifidobacterium bifidum DSM20215

SEQ ID NO: 9 is nucleotide sequence encoding BIF_(—)917

SEQ ID NO: 10 is nucleotide sequence encoding BIF_(—)995

SEQ ID NO: 11 is nucleotide sequence encoding BIF_(—)1068

SEQ ID NO: 12 is nucleotide sequence encoding BIF_(—)1172

SEQ ID NO: 13 is nucleotide sequence encoding BIF_(—)1241

SEQ ID NO: 14 is nucleotide sequence encoding BIF_(—)1326

SEQ ID NO: 15 is forward primer for generation of above BIF variants

SEQ ID NO: 16 is reverse primer for BIF917

SEQ ID NO: 17 is reverse primer for BIF995

SEQ ID NO: 18 is reverse primer for BIF1068

SEQ ID NO: 19 is reverse primer for BIF1241

SEQ ID NO: 20 is reverse primer for BIF1326

SEQ ID NO: 21 is reverse primer for BIF1478

SEQ ID NO: 22 is extracellular lactase from Bifidobacterium bifidumDSM20215.

SEQ ID NO: 23 is signal sequence of extracellular lactase fromBifidobacterium bifidum DSM20215

DETAILED DISCLOSURE OF THE INVENTION Definitions

In accordance with this detailed description, the followingabbreviations and definitions apply. It should be noted that as usedherein, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a polypeptide” includes a plurality of such polypeptides,and reference to “the formulation” includes reference to one or moreformulations and equivalents thereof known to those skilled in the art,and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. The following terms are provided below.

“Transgalactosylase” means an enzyme that, among other things, is ableto transfer galactose to the hydroxyl groups of D-galactose or D-glucosewhereby galacto-oligosaccharides are produced. In one aspect, atransgalactosylase is identified by reaction of the enzyme on lactose inwhich the amount of galactose generated is less than the amount ofglucose generated at any given time.

In the present context, the term “transgalactosylating activity” meansthe transfer of a galactose moiety to a molecule other than water. Theactivity can be measured as [glucose]-[galactose] generated at any giventime during reaction or by direct quantification of the GOS generated atany given time during the reaction. This measurement may be performed inseveral ways such as by a HPLC method as shown in the examples. Whencomparing measurements of transgalactosylating activity, they have beenperformed at a given initial lactose concentration, such as e.g. 3, 4,5, 6, 7, 8, 9 or 10% (w/w).

In the present context, the term “β-galactosidase activity” means theability of an enzyme to hydrolyse β-galactosides such as for examplelactose into monosaccharides, glucose and galactose.

In the context of calculating transgalactosylatingactivity:β-galactosidase activity, the β-galactosidase activity ismeasured as [galactose] generated at any given time during reaction.This measurement may be performed in several ways such as by a HPLCmethod as shown in the examples.

In the present context, the term “ratio of transgalactosylationactivity” using ortho-nitrophenol-β-D-galactopyranoside (ONPG) wascalculated as follows: Ratio is calculated as ratio between Abs420 withacceptor present divided by Abs420 without acceptor present times 100.Variant at or below index 100 are purely hydrolytic variants, whereasthe level above depicts relative transgalactosylating activity.

Ratio of transgalactosylationactivity=(Abs420^(+Cellobiose)/Abs420^(−Cellobiose))*100%, whereAbs420^(+Cellobiose) is the absorbance read at 420 nm using thedescribed method 3 below including cellobiose in the reaction andAbs420^(−Cellobiose) is the absorbance read at 420 nm using thedescribed method 3 below but without cellobiose in the reaction. Theequation above is only valid for dilutions where the absorbance isbetween 0.5 and 1.0.

In one aspect, the activity is measured after 15 min. reaction, 30 min.reaction, 60 min. reaction, 90 min. reaction, 120 min. reaction or 180min. reaction. Thus in one aspect, as an example the relativetransgalactosylation activity is measured 15 minutes after addition ofenzyme, such as 30 minutes after addition of enzyme, such as 60 minutesafter addition of enzyme, such as 90 minutes after addition of enzyme,such as 120 minutes after addition of enzyme or such as 180 minutesafter addition of enzyme.

In the present context, the term “ratio of transgalactosylatingactivity:β-galactosidase activity” means([Glucose]−[Galactose]/[Galactose]).

In the present context, the term [Glucose] means the glucoseconcentration in % by weight as measured by HPLC.

In the present context, the term [Galactose] means the galactoseconcentration in % by weight as measured by HPLC.

In the present context, the term “lactose has been transgalactosylated”means that a galactose molecule has been covalently linked to thelactose molecule such as for example covalently linked to any of thefree hydroxyl groups in the lactose molecule or as generated by internaltransgalatosylation for example forming allolactose.

In the present context, the evaluation of performance of polypeptidesdisclosed herein in galactooligosaccharide (GOS) production were testedin a “milk-based assay” (yogurt application mimic). Batch experimentswith a volume of 100 μl were performed in 96 well MTP plates using ayogurt mix, consisting of 98.60%(w/v) fresh pasteurized low-fat milk(Arla Mini-mælk) and 1.4% (w/v) Nutrilac YQ-5075 whey ingredient (Arla).To completely hydrate Nutrilac YQ-5075 the mixture was left withagitation for 20 h and afterwards added 20 mM NaPhosphate pH 6.5 toensure a pH of 6.5. This yogurt-base was either used plain or withvarious supplements such as additional lactose, fucose, maltose, xyloseor salts. 90 μl of the yogurt was mixed with 10 μl purified enzyme orcrude ferment, sealed with tape and incubated at 43° C. for 3 hours. Thereaction was stopped by 100 μl 10 Na2CO3. Samples were stored at −20° C.Quantification of galactooligosaccharides (GOS), lactose, glucose andgalactose were performed by HPLC. Analysis of samples was carried out ona Dionex ICS 3000. IC parameters were as follows: Mobile phase: 150 mMNaOH, Flow: Isochratic, 0.25 ml/min, Column: Carbopac PA1, Columntemperature: RT, Injection volume: 10 μL, Detector: PAD, Integration:Manual, Sample preparation: 100 times dilution in Milli-Q water (0.1 mlsample+9.9 ml water) and filtration through 0.45 ìm syringe filters,Quantification: Peak areas in percent of peak area of the standard. AGOS syrup (Vivanal GOS, Friesland Campina) was used as standard for GOSquantification. Results of such an evaluation is shown in FIG. 4, andfurther described in example 2.

In the present context, the term “which polypeptide is freeze-dried”means that the polypeptide has been obtained by freeze-drying a liquidof the polypeptide at an appropriate pressure and for an appropriateperiod removing the water.

In the present context, the term “which polypeptide is in solution”relates to a polypeptide which is soluble in a solvent withoutprecipitating out of solution. A solvent for this purpose includes anymillieu in which the polypeptide may occur, such as an aqueous buffer orsalt solution, a fermentation broth, or the cytoplasm of an expressionhost.

In the present context, the term “stabilizer” means any stabilizer forstabilizing the polypeptide e.g., a polyol such as, e.g., glycerol orpropylene glycol, a sugar or a sugar alcohol, lactic acid, boric acid,or a boric acid derivative (e.g., an aromatic borate ester). In oneaspect, the stabilizer is glycerol.

In the present context, the term “carbohydrate substrate” means anorganic compound with the general formula C_(m)(H₂O)_(n), that is,consisting only of carbon, hydrogen and oxygen, the last two in the 2:1atom ratio such as a disaccharide.

In the present context, the term “disaccharide” is two monosaccharideunits bound together by a covalent bond known as a glycosidic linkageformed via a dehydration reaction, resulting in the loss of a hydrogenatom from one monosaccharide and a hydroxyl group from the other. Theformula of unmodified disaccharides is C₁₂H₂₂O₁₁. In one aspect, thedisaccharide is lactulose, trehalose, rhamnose, maltose, sucrose,lactose, fucose or cellobiose. In a further aspect, the disaccharide islactose.

The term “isolated” means that the polypeptide is at least substantiallyfree from at least one other component with which the sequence isnaturally associated in nature and as found in nature. In one aspect,“isolated polypeptide” as used herein refers to a polypeptide which isat least 30% pure, at least 40% pure, at least 60% pure, at least 80%pure, at least 90% pure, and at least 95% pure, as determined bySDS-PAGE.

The term “substantially pure polypeptide” means herein a polypeptidepreparation which contains at most 10%, preferably at most 8%, morepreferably at most 6%, more preferably at most 5%, more preferably atmost 4%, at most 3%, even more preferably at most 2%, most preferably atmost 1%, and even most preferably at most 0.5% by weight of otherpolypeptide material with which it is natively associated. It is,therefore, preferred that the substantially pure polypeptide is at least92% pure, preferably at least 94% pure, more preferably at least 95%pure, more preferably at least 96% pure, more preferably at least 96%pure, more preferably at least 97% pure, more preferably at least 98%pure, even more preferably at least 99%, most preferably at least 99.5%pure, and even most preferably 100% pure by weight of the totalpolypeptide material present in the preparation. The polypeptidesdisclosed herein are preferably in a substantially pure form. Inparticular, it is preferred that the polypeptides are in “essentiallypure form”, i.e., that the polypeptide preparation is essentially freeof other polypeptide material with which it is natively associated. Thiscan be accomplished, for example, by preparing the polypeptide by meansof well-known recombinant methods or by classical purification methods.Herein, the term “substantially pure polypeptide” is synonymous with theterms “isolated polypeptide” and “polypeptide in isolated form.”

The term “purified” or “pure” means that a given component is present ata high level state—e.g. at least about 51% pure, such as at least 51%pure, or at least about 75% pure such as at least 75% pure, or at leastabout 80% pure such as at least 80% pure, or at least about 90% puresuch as at least 90% pure, or at least about 95% pure such as at least95% pure, or at least about 98% pure such as at least 98% pure. Thecomponent is desirably the predominant active component present in acomposition.

The term “microorganism” in relation to the present invention includesany “microorganism” that could comprise a nucleotide sequence accordingto the present invention or a nucleotide sequence encoding for apolypeptide having the specific properties as defined herein and/orproducts obtained therefrom. In the present context, “microorganism” mayinclude any bacterium or fungus being able to ferment a milk substrate.

The term “host cell”—in relation to the present invention includes anycell that comprises either a nucleotide sequence encoding a polypeptidehaving the specific properties as defined herein or an expression vectoras described above and which is used in the production of a polypeptidehaving the specific properties as defined herein. In one aspect, theproduction is recombinant production.

The term “milk”, in the context of the present invention, is to beunderstood as the lacteal secretion obtained from any mammal, such ascows, sheep, goats, buffaloes or camels. In the present context, theterm “milk-based substrate” means any raw and/or processed milk materialor a material derived from milk constituents. Useful milk-basedsubstrates include, but are not limited to solutions/suspensions of anymilk or milk like products comprising lactose, such as whole or low fatmilk, skim milk, buttermilk, reconstituted milk powder, condensed milk,solutions of dried milk, UHT milk, whey, whey permeate, acid whey, orcream. Preferably, the milk-based substrate is milk or an aqueoussolution of skim milk powder. The milk-based substrate may be moreconcentrated than raw milk. In one embodiment, the milk-based substratehas a ratio of protein to lactose of at least 0.2, preferably at least0.3, at least 0.4, at least 0.5, at least 0.6 or, most preferably, atleast 0.7.

The milk-based substrate may be homogenized and/or pasteurized accordingto methods known in the art.

“Homogenizing” as used herein means intensive mixing to obtain a solublesuspension or emulsion. It may be performed so as to break up the milkfat into smaller sizes so that it no longer separates from the milk.This may be accomplished by forcing the milk at high pressure throughsmall orifices.

“Pasteurizing” as used herein means reducing or eliminating the presenceof live organisms, such as microorganisms, in the milk-based substrate.Preferably, pasteurization is attained by maintaining a specifiedtemperature for a specified period of time. The specified temperature isusually attained by heating. The temperature and duration may beselected in order to kill or inactivate certain bacteria, such asharmful bacteria, and/or to inactivate enzymes in the milk. A rapidcooling step may follow. A “food product” or “food composition” in thecontext of the present invention may be any comestible food or feedproduct suitable for consumption by an animal or human.

A “dairy product” in the context of the present invention may be anyfood product wherein one of the major constituents is milk-based.Preferable, the major constituent is milk-based. More preferably, themajor constituent is a milk-based substrate which has been treated withan enzyme having transgalactosylating activity.

In the present context, “one of the major constituents” means aconstituent having a dry matter which constitutes more than 20%,preferably more than 30% or more than 40% of the total dry matter of thedairy product, whereas “the major constituent” means a constituenthaving a dry matter which constitutes more than 50%, preferably morethan 60% or more than 70% of the total dry matter of the dairy product.

A “fermented dairy product” in present context is to be understood asany dairy product wherein any type of fermentation forms part of theproduction process. Examples of fermented dairy products are productslike yoghurt, buttermilk, creme fraiche, quark and fromage frais.Another example of a fermented dairy product is cheese. A fermenteddairy product may be produced by any method known in the art.

The term “fermentation” means the conversion of carbohydrates intoalcohols or acids through the action of a microorganism such as astarter culture. In one aspect, fermentation comprises conversion oflactose to lactic acid.

In the present context, “microorganism” may include any bacterium orfungus being able to ferment a milk substrate.

In the present context the term “Pfam domains” means regions within aprotein sequence that are identified as either Pfam-A or Pfam-B based onmultiple sequence alignments and the presence of Hidden Markov Motifs(“The Pfam protein families database”: R. D. Finn, J. Mistry, J. Tate,P. Coggill, A. Heger, J. E. Pollington, O. L. Gavin, P. Gunesekaran, G.Ceric, K. Forslund, L. Holm, E. L. Sonnhammer, S. R. Eddy, A. BatemanNucleic Acids Research (2010) Database Issue 38:D211-222.). As examplesof Pfam domains mention may be made of Glyco_hydro2N (PF02837),Glyco_hydro (PF00703), Glyco_hydro 2C (PF02836) and Bacterial Ig-likedomain (group 4) (PF07532).

As used herein “a position corresponding to position” means that analignment as described herein is made between a particular querypolypeptide and the reference polypeptide. The position corresponding toa specific position in the reference polypeptide is then identified asthe corresponding amino acid in the alignment with the highest sequenceidentity.

A “variant” or “variants” refers to either polypeptides or nucleicacids. The term “variant” may be used interchangeably with the term“mutant”. Variants include insertions, substitutions, transversions,truncations, and/or inversions at one or more locations in the aminoacid or nucleotide sequence, respectively. The phrases “variantpolypeptide”, “polypeptide variant”, “polypeptide”, “variant” and“variant enzyme” mean a polypeptide/protein that has an amino acidsequence that either has or comprises a selected amino acid sequence ofor is modified compared to the selected amino acid sequence, such as SEQID NO: 1, 2, 3, 4 or 5.

As used herein, “reference enzymes,” “reference sequence,” “referencepolypeptide” mean enzymes and polypeptides from which any of the variantpolypeptides are based, e.g., SEQ ID NO: 1, 2, 3, 4 or 5. A “referencenucleic acid” means a nucleic acid sequence encoding the referencepolypeptide.

As used herein, the terms “reference sequence” and “subject sequence”are used interchangeably.

As used herein, “query sequence” means a foreign sequence, which isaligned with a reference sequence in order to see if it falls within thescope of the present invention. Accordingly, such query sequence can forexample be a prior art sequence or a third party sequence.

As used herein, the term “sequence” can either be referring to apolypeptide sequence or a nucleic acid sequence, depending of thecontext.

As used herein, the terms “polypeptide sequence” and “amino acidsequence” are used interchangeably.

The signal sequence of a “variant” may be the same or may differ fromthe signal sequence of the wild-type a Bacillus signal peptide or anysignal sequence that will secrete the polypeptide. A variant may beexpressed as a fusion protein containing a heterologous polypeptide. Forexample, the variant can comprise a signal peptide of another protein ora sequence designed to aid identification or purification of theexpressed fusion protein, such as a His-Tag sequence.

To describe the various variants that are contemplated to be encompassedby the present disclosure, the following nomenclature will be adoptedfor ease of reference. Where the substitution includes a number and aletter, e.g., 592P, then this refers to {position according to thenumbering system/substituted amino acid}. Accordingly, for example, thesubstitution of an amino acid to proline in position592 is designated as592P. Where the substitution includes a letter, a number, and a letter,e.g., D592P, then this refers to {original amino acid/position accordingto the numbering system/substituted amino acid}.

Accordingly, for example, the substitution of alanine with proline inposition 592 is designated as A592P.

Where two or more substitutions are possible at a particular position,this will be designated by contiguous letters, which may optionally beseparated by slash marks “/”, e.g., G303ED or G303E/D.

Position(s) and substitutions are listed with reference to for exampleeither SEQ ID NO: 1, 2, 3, 4 or 5. For example equivalent positions inanother sequence may be found by aligning this sequence with either SEQID NO: 1, 2, 3, 4 or 5 to find an alignment with the highest percentidentity and thereafter determining which amino acid aligns tocorrespond with an amino acid of a specific position of either SEQ IDNO: 1, 2, 3, 4 or 5. Such alignment and use of one sequence as a firstreference is simply a matter of routine for one of ordinary skill in theart.

As used herein, the term “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation.

As used herein, “polypeptide” is used interchangeably with the terms“amino acid sequence”, “enzyme”, “peptide” and/or “protein”. As usedherein, “nucleotide sequence” or “nucleic acid sequence” refers to anoligonucleotide sequence or polynucleotide sequence and variants,homologues, fragments and derivatives thereof. The nucleotide sequencemay be of genomic, synthetic or recombinant origin and may bedouble-stranded or single-stranded, whether representing the sense oranti-sense strand. As used herein, the term “nucleotide sequence”includes genomic DNA, cDNA, synthetic DNA, and RNA.

“Homologue” means an entity having a certain degree of identity or“homology” with the subject amino acid sequences and the subjectnucleotide sequences. In one aspect, the subject amino acid sequence isSEQ ID NO: 1, 2, 3, 4 or 5, and the subject nucleotide sequencepreferably is SEQ ID NO: 9, 10, 11, 12 or 13.

A “homologous sequence” includes a polynucleotide or a polypeptidehaving a certain percent, e.g., 80%, 85%, 90%, 95%, or 99%, of sequenceidentity with another sequence. Percent identity means that, whenaligned, that percentage of bases or amino acid residues are the samewhen comparing the two sequences. Amino acid sequences are notidentical, where an amino acid is substituted, deleted, or addedcompared to the subject sequence. The percent sequence identitytypically is measured with respect to the mature sequence of the subjectprotein, i.e., following removal of a signal sequence, for example.Typically, homologues will comprise the same active site residues as thesubject amino acid sequence. Homologues also retain enzymatic activity,although the homologue may have different enzymatic properties than thewild-type.

As used herein, “hybridization” includes the process by which a strandof nucleic acid joins with a complementary strand through base pairing,as well as the process of amplification as carried out in polymerasechain reaction (PCR) technologies. The variant nucleic acid may exist assingle- or double-stranded DNA or RNA, an RNA/DNA heteroduplex or anRNA/DNA copolymer. As used herein, “copolymer” refers to a singlenucleic acid strand that comprises both ribonucleotides anddeoxyribonucleotides. The variant nucleic acid may be codon-optimized tofurther increase expression.

As used herein, a “synthetic” compound is produced by in vitro chemicalor enzymatic synthesis. It includes, but is not limited to, variantnucleic acids made with optimal codon usage for host organisms, such asa yeast cell host or other expression hosts of choice.

As used herein, “transformed cell” includes cells, including bothbacterial and fungal cells, which have been transformed by use ofrecombinant DNA techniques. Transformation typically occurs by insertionof one or more nucleotide sequences into a cell. The inserted nucleotidesequence may be a heterologous nucleotide sequence, i.e., is a sequencethat is not natural to the cell that is to be transformed, such as afusion protein.

As used herein, “operably linked” means that the described componentsare in a relationship permitting them to function in their intendedmanner. For example, a regulatory sequence operably linked to a codingsequence is ligated in such a way that expression of the coding sequenceis achieved under condition compatible with the control sequences.

As used herein, the term “fragment” is defined herein as a polypeptidehaving one or more (several) amino acids deleted from the amino and/orcarboxyl terminus wherein the fragment has activity.

In one aspect, the term “fragment” is defined herein as a polypeptidehaving one or more (several) amino acids deleted from the amino and/orcarboxyl terminus of the polypeptide of SEQ ID NO: 1, 2, 3, 4 or 5;wherein the fragment has transgalactosylating activity.

The term “Galactose Binding domain-like” as used herein is abbreviatedto and interchangeable with the term “GBD”.

Degree of Identity

The relatedness between two amino acid sequences or between twonucleotide sequences is described by the parameter “identity”.

In one embodiment, the degree of sequence identity between a querysequence and a reference sequence is determined by 1) aligning the twosequences by any suitable alignment program using the default scoringmatrix and default gap penalty, 2) identifying the number of exactmatches, where an exact match is where the alignment program hasidentified an identical amino acid or nucleotide in the two alignedsequences on a given position in the alignment and 3) dividing thenumber of exact matches with the length of the reference sequence.

In one embodiment, the degree of sequence identity between a querysequence and a reference sequence is determined by 1) aligning the twosequences by any suitable alignment program using the default scoringmatrix and default gap penalty, 2) identifying the number of exactmatches, where an exact match is where the alignment program hasidentified an identical amino acid or nucleotide in the two alignedsequences on a given position in the alignment and 3) dividing thenumber of exact matches with the length of the longest of the twosequences.

In another embodiment, the degree of sequence identity between the querysequence and the reference sequence is determined by 1) aligning the twosequences by any suitable alignment program using the default scoringmatrix and default gap penalty, 2) identifying the number of exactmatches, where an exact match is where the alignment program hasidentified an identical amino acid or nucleotide in the two alignedsequences on a given position in the alignment and 3) dividing thenumber of exact matches with the “alignment length”, where the alignmentlength is the length of the entire alignment including gaps andoverhanging parts of the sequences.

Sequence identity comparisons can be conducted by eye, or more usually,with the aid of readily available sequence comparison programs. Thesecommercially available computer programs use complex comparisonalgorithms to align two or more sequences that best reflect theevolutionary events that might have led to the difference(s) between thetwo or more sequences. Therefore, these algorithms operate with ascoring system rewarding alignment of identical or similar amino acidsand penalising the insertion of gaps, gap extensions and alignment ofnon-similar amino acids. The scoring system of the comparison algorithmsinclude:

-   -   i) assignment of a penalty score each time a gap is inserted        (gap penalty score),    -   ii) assignment of a penalty score each time an existing gap is        extended with an extra position (extension penalty score),    -   iii) assignment of high scores upon alignment of identical amino        acids, and    -   iv) assignment of variable scores upon alignment of        non-identical amino acids.

Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons.

The scores given for alignment of non-identical amino acids are assignedaccording to a scoring matrix also called a substitution matrix. Thescores provided in such substitution matrices are reflecting the factthat the likelihood of one amino acid being substituted with anotherduring evolution varies and depends on the physical/chemical nature ofthe amino acid to be substituted. For example, the likelihood of a polaramino acid being substituted with another polar amino acid is highercompared to being substituted with a hydrophobic amino acid. Therefore,the scoring matrix will assign the highest score for identical aminoacids, lower score for non-identical but similar amino acids and evenlower score for non-identical non-similar amino acids. The mostfrequently used scoring matrices are the PAM matrices (Dayhoff et al.(1978), Jones et al. (1992)), the BLOSUM matrices (Henikoff and Henikoff(1992)) and the Gonnet matrix (Gonnet et al. (1992)).

Suitable computer programs for carrying out such an alignment include,but are not limited to, Vector NTI (Invitrogen Corp.) and the ClustalV,ClustalW and ClustalW2 programs (Higgins D G & Sharp P M (1988), Higginset al. (1992), Thompson et al. (1994), Larkin et al. (2007). A selectionof different alignment tools is available from the ExPASy Proteomicsserver at www.expasy.org. Another example of software that can performsequence alignment is BLAST (Basic Local Alignment Search Tool), whichis available from the webpage of National Center for BiotechnologyInformation which can currently be found at http://www.ncbi.nlm.nih.gov/and which was firstly described in Altschul et al. (1990) J. Mol. Biol.215; 403-410.

In a preferred embodiment of the present invention, the alignmentprogram is performing a global alignment program, which optimizes thealignment over the full-length of the sequences. In a further preferredembodiment, the global alignment program is based on theNeedleman-Wunsch algorithm (Needleman, Saul B.; and Wunsch, Christian D.(1970), “A general method applicable to the search for similarities inthe amino acid sequence of two proteins”, Journal of Molecular Biology48 (3): 443-53). Examples of current programs performing globalalignments using the Needleman-Wunsch algorithm are EMBOSS Needle andEMBOSS Stretcher programs, which are both available athttp://www.ebi.ac.uk/Tools/psa/.

EMBOSS Needle performs an optimal global sequence alignment using theNeedleman-Wunsch alignment algorithm to find the optimum alignment(including gaps) of two sequences along their entire length.

EMBOSS Stretcher uses a modification of the Needleman-Wunsch algorithmthat allows larger sequences to be globally aligned.

In one embodiment, the sequences are aligned by a global alignmentprogram and the sequence identity is calculated by identifying thenumber of exact matches identified by the program divided by the“alignment length”, where the alignment length is the length of theentire alignment including gaps and overhanging parts of the sequences.

In a further embodiment, the global alignment program uses theNeedleman-Wunsch algorithm and the sequence identity is calculated byidentifying the number of exact matches identified by the programdivided by the “alignment length”, where the alignment length is thelength of the entire alignment including gaps and overhanging parts ofthe sequences.

In yet a further embodiment, the global alignment program is selectedfrom the group consisting of EMBOSS Needle and EMBOSS stretcher and thesequence identity is calculated by identifying the number of exactmatches identified by the program divided by the “alignment length”,where the alignment length is the length of the entire alignmentincluding gaps and overhanging parts of the sequences.

Once the software has produced an alignment, it is possible to calculate% similarity and ° A) sequence identity. The software typically doesthis as part of the sequence comparison and generates a numericalresult.

In one embodiment, it is preferred to use the ClustalW software forperforming sequence alignments. Preferably, alignment with ClustalW isperformed with the following parameters for pairwise alignment:

Substitution matrix: Gonnet 250 Gap open penalty: 20 Gap extensionpenalty: 0.2 Gap end penalty: None

ClustalW2 is for example made available on the internet by the EuropeanBioinformatics Institute at the EMBL-EBI webpage www.ebi.ac.uk undertools—sequence analysis—ClustalW2. Currently, the exact address of theClustalW2 tool is www.ebi.ac.uk/Tools/clustalw2.

In another embodiment, it is preferred to use the program Align X inVector NTI (Invitrogen) for performing sequence alignments. In oneembodiment, Exp10 has been may be used with default settings:

Gap opening penalty: 10Gap extension penalty: 0.05Gap separation penalty range: 8

In a another embodiment, the alignment of one amino acid sequence with,or to, another amino acid sequence is determined by the use of the scorematrix: blosum62mt2 and the VectorNTI Pair wise alignment settings

Settings K-tuple 1 Number of best diagonals 5 Window size 5 Gap Penalty3 Gap opening Penalty 10 Gap extension Penalty 0.1

In one embodiment, the percentage of identity of one amino acid sequencewith, or to, another amino acid sequence is determined by the use ofBlast with a word size of 3 and with BLOSUM 62 as the substitutionmatrix

Polypeptides

In one aspect, disclosed herein is a polypeptide having a ratio oftransgalactosylating activity:β-galactosidase activity of at least 0.5,at least 1, at least 2, at least 2.5, at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, at least 10, at least11, or at least 12 at or above a concentration of 3% w/w initial lactoseconcentration.

In one aspect, disclosed herein is a polypeptide, wherein the glycosidehydrolase catalytic core has an amino acid sequence of SEQ ID NO:7.

In one aspect, disclosed herein is a polypeptide containing aGlyco_hydro2N (PF02837), a Glyco_hydro (PF00703) and/or a Glyco_hydro 2C(PF02836) domains.

In one aspect, disclosed herein is a polypeptide containing theBacterial Ig-like domain (group 4) (PF07532).

In one aspect, disclosed herein is a polypeptide havingtransgalactosylating activity selected from the group consisting of:

-   -   a. a polypeptide comprising an amino acid sequence having at        least 90% sequence identity with SEQ ID NO: 1, wherein said        polypeptide consists of at most 980 amino acid residues,    -   b. a polypeptide comprising an amino acid sequence having at        least 97% sequence identity with SEQ ID NO: 2, wherein said        polypeptide consists of at most 975 amino acid residues,    -   c. a polypeptide comprising an amino acid sequence having at        least 96.5% sequence identity with SEQ ID NO: 3, wherein said        polypeptide consists of at most 1300 amino acid residues,    -   d. a polypeptide encoded by a polynucleotide that hybridizes        under at least low stringency conditions with i) the nucleic        acid sequence comprised in SEQ ID NO: 9, 10, 11, 12 or 13        encoding the polypeptide of SEQ ID NO: 1, 2, 3, 4 or 5; or ii)        the complementary strand of i),    -   e. a polypeptide encoded by a polynucleotide comprising a        nucleotide sequence having at least 70% identity to the        nucleotide sequence encoding for the polypeptide of SEQ ID NO:        1, 2, 3, 4 or 5 or the nucleotide sequence comprised in SEQ ID        NO: 9, 10, 11, 12 or 13 encoding a mature polypeptide, and    -   f. a polypeptide comprising a deletion, insertion and/or        conservative substitution of one or more amino acid residues of        SEQ ID NO: 1, 2, 3, 4 or 5.

In another aspect disclosed herein is a polypeptide havingtransgalactosylating activity selected from the group consisting of:

-   -   a. a polypeptide comprising an amino acid sequence having at        least 96.5% sequence identity with SEQ ID NO: 3, wherein said        polypeptide consists of at most 1300 amino acid residues,    -   b. a polypeptide comprising an amino acid sequence having at        least 90% sequence identity with SEQ ID NO: 1, wherein said        polypeptide consists of at most 980 amino acid residues,    -   c. a polypeptide encoded by a polynucleotide that hybridizes        under at least low stringency conditions with i) the nucleic        acid sequence comprised in SEQ ID NO: 9, 10, 11, 12 or 13        encoding the polypeptide of SEQ ID NO: 1, 2, 3, 4, or 5; or ii)        the complementary strand of i),    -   d. a polypeptide encoded by a polynucleotide comprising a        nucleotide sequence having at least 70% identity to the        nucleotide sequence encoding for the polypeptide of SEQ ID NO:        1, 2, 3, 4 or 5 or the nucleotide sequence comprised in SEQ ID        NO: 9, 10, 11, 12 or 13 encoding a mature polypeptide, and    -   e. a polypeptide comprising a deletion, insertion and/or        conservative substitution of one or more amino acid residues of        SEQ ID NO: 1, 2, 3, 4 or 5.

In one aspect, disclosed herein is a polypeptide, wherein the amino acidsequence has at least 68%, 70%, 72%, 74%, 76%, 78%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to themature amino acid sequence of SEQ ID NO: 1, 2, 3, 4 or 5.

In one aspect, disclosed herein is a polypeptide having 90% sequenceidentity to the mature amino acid sequence of SEQ ID NO:1.

In one aspect, disclosed herein is a polypeptide having 90% sequenceidentity to the mature amino acid sequence of SEQ ID NO:2.

In one aspect, disclosed herein is a polypeptide having 96.5% sequenceidentity to the mature amino acid sequence of SEQ ID NO:3.

In one aspect, disclosed herein is a polypeptide having 96.5% sequenceidentity to the mature amino acid sequence of SEQ ID NO:4.

In one aspect, disclosed herein is a polypeptide having 96.5% sequenceidentity to the mature amino acid sequence of SEQ ID NO:5.

In one aspect, disclosed herein is a polypeptide comprising orconsisting of the amino acid sequence of SEQ ID NO:1, 2, 3, 4 or 5.

In one aspect, disclosed herein is a polypeptide, which is derived fromBifidobacterium bifidum.

In one aspect, disclosed herein is a polypeptide having a pH optimum of6.5-7.5.

In one aspect, disclosed herein is a polypeptide having a temperatureoptimum of 30-60 such as 42-60 degree celcius.

Polypeptides having activity on carbohydrates can be classified usingeither the IUBMB system of classification based on their substratespecificity or on the CaZy assignment into one of the current 125glycoside hydrolase family. In the CaZy database the assignment is basedon both sequence and structural information combined with knowledge ofstereochemistry of the substrates and products

Disclosed herein are polypeptides which when being an expression productin a Bacillus subtilis strain BG3594 of a nucleic acid sequence, whichencodes said polypeptide, is the only polypeptide expression product ofsaid nucleic acid sequence that exhibits transgalactosylating activity.This may be evaluated by using the following techniques know to a personskilled in the art. The samples to be evaluated are subjected toSDS-PAGE and visualized using a dye appropriate for proteinquantification, such as for example the Bio-Rad Criterion system. Thegel is then scanned using appropriate densiometic scanner such as forexample the Bio-Rad Criterion system and the resulting picture isensured to be in the dynamic range. The bands corresponding to anyvariant/fragment derived from SEQ ID NO: 8 are quantified and thepercentage of the polypeptides are calculated as: Percentage ofpolypeptide in question=polypeptide in question/(sum of all polypeptidesexhibiting transgalactosylating activity)*100.

The total number of polypeptides variants/fragments derived from SEQ IDNO:8 in the composition can be determined by detecting fragment derivedfrom SEQ ID NO:8 by western blotting using a polyclonal antibody bymethods know to a person skilled in the art.

The polypeptide disclosed herein comprises at least two separatefunctional domains contained within the enzyme. Firstly, the polypeptideshould contain a glycoside hydrolase catalytic core as described in thefollowing. The catalytic core should belong to the GH-A clan of relatedglycoside hydrolase families. The GH-A clan is characterized by cleavingglycosidic bonds via a retaining mechanism and possesses a catalyticdomain which is based on a TIM barrel fold (Wierenga, 2001, FEBSLetters, 492(3), p 193-8). The catalytic domain contains two glutamicacid residues which act as proton donor and nucleophile, emanating fromstrands 4 and 7 of the barrel domain (Jenkins, 1995, FEBS Letters,362(3), p 281-5). The overall structure of the TIM barrel is a (β/α) 8fold consisting of 8 beta strands and 8 alpha-helices. In one aspect,the glycoside hydrolase catalytic core disclosed herein belong to eitherof the glycoside hydrolase families GH-2, and -35 which are allTIM-barrel enzymes belonging to the GH-A clan. In a further aspect, theglycoside hydrolase catalytic core belong to family GH-2 or GH-35. In afurther aspect, the glycoside hydrolase catalytic core belong to familyGH-2. A common denominator is that these enzymes are so called retainingenzymes, so that the stereochemistry of the substrate is conserved inthe product (Henrissat, 1997, Curr Opin Struct Biol, 7(5), 637-44).

In one aspect, the polypeptides disclosed herein have activity oncarbohydrates bonds which has the β(1→4) conformation. This effectivelyput the enzymes into the IUBMB EC 3.2.1.23 class of β-galactosidases.This activity may be, but is not confined to, determined by utilizingsynthetic substrates such as para-nitrophenol-β-D-galactopyranoside(PNPG), ortho-nitrophenol-β-D-galactopyranoside (ONPG) orβ-D-galactopyranoside with chromogenic aglycons (XGaI). As analternative way of determining whether an enzyme belong to the EC3.2.1.23 class of β-galactosidases is to incubate with a substrate suchas lactose and measure the release of glucose by a method such asenzymatic determination, HPLC, TLC or other methods known to personsskilled in the art.

In order to predict functional entities of polypeptides severalavailable public repositories can be applied such as for example Pfam(Nucl. Acids Res. (2010) 38 (suppl 1): D211-D222. doi:10.1093/nar/gkp985) and Interpro (Nucl. Acids Res. (2009) 37 (suppl 1):D211-D215. doi: 10.1093/nar/gkn785). It should be specified that whenperforming such analysis the analysis should be performed on the fulllength sequence of the polypeptide available from public repositorydatabases.

In a further aspect, a polypeptide containing one or more Pfam domainsselected from: Glyco_hydro2N (PF02837), Glyco_hydro (PF00703),Glyco_hydro 2C (PF02836) and Bacterial Ig-like domain (group 4)(PF07532), is provided. In yet a further aspect, a polypeptidecontaining the Pfam domains Glyco_hydro2N (PF02837), Glyco_hydro(PF00703), Glyco_hydro 2C (PF02836) and Bacterial Ig-like domain (group4) (PF07532), is provided. In yet a further aspect, a polypeptidecontaining the Glyco_hydro2N (PF02837), Glyco_hydro (PF00703), andGlyco_hydro 2C (PF02836) domains which constitutes the catalytic domainof the polypeptide, is provided.

In a further aspect, a polypeptide as disclosed herein and having aratio of transgalactosylating activity:β-galactosidase activity of atleast 1, at least 2.5, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, or atleast 12 as measured at a concentration of 100 ppm in a milk-based assayat 37° C. and 5 w/w % lactose after 15, 30 or 180 such as 180 minutesreaction, is provided. In a further aspect, the polypeptide is derivedfrom Bifidobacterium bifidum.

In one aspect, the herein disclosed polypeptide(s) has atransgalactosylating activity such that more than 20%, more than 30%,more than 40%, up to 50% of the initial lactose is transgalactosylatedas measured at a concentration of 100 ppm in a milk-based assay at 37°C. and 5 w/w % lactose after 15, 30 or 180 such as 180 minutes ofreaction.

In a further aspect, the herein disclosed polypeptide(s) has aβ-galactosidase activity such that less than 80%, less than 70%, lessthan 60%, less than 50%, less than 40%, less than 30%, less than 20% ofthe lactose has been hydrolysed as measured at a concentration of 100ppm in a milk-based assay at 37° C. and 5 w/w % lactose after 15, 30 or180 such as 180 minutes of reaction.

In one aspect, the β-galactosidase activity and/or thetransgalactosylating activity are measured at a concentration of 100 ppmcorresponding to 2.13 LAU as specified in method 4.

In a further aspect, the herein disclosed polypeptide(s) has one or moreof the following characteristics:

a) a ratio of transgalactosylating activity:β-galactosidase activity ofat least of at least 1, at least 2.5, at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, at least 10, at least11, or at least 12 as measured at a concentration of 100 ppm in amilk-based assay at 37° C. and 5 w/w % lactose after 15, 30 or 180 suchas 180 minutes reaction, and/orb) has a transgalactosylating activity such that more than 20%, morethan 30%, more than 40%, and up to 50% of the initial lactose has beentransgalactosylated as measured at a concentration of 100 ppm in amilk-based assay at 37° C. and 5 w/w % lactose after 15, 30 or 180 suchas 180 minutes of reaction.

In one aspect, a polypeptide comprising an amino acid sequence having atleast 96.5% sequence identity with SEQ ID NO: 3, wherein saidpolypeptide consists of at most 1300 amino acid residues, is provided.In a further aspect, a polypeptide comprising an amino acid sequencehaving at least 90% sequence identity with SEQ ID NO: 1 such as whereinsaid sequence identity is at least 95%, such as, e.g. at least 96%, atleast 97%, at least 98%, at least 99% or at least 100% sequenceidentity, and wherein said polypeptide consists of at most 980 aminoacid residues, is provided. In a further aspect, a polypeptidecomprising an amino acid sequence having at least 90% sequence identitywith SEQ ID NO: 1, wherein said polypeptide consists of at most 980amino acid residues, is provided. In yet a further aspect, a polypeptidewherein said polypeptide has at least 90% sequence identity with SEQ IDNO: 1, such as wherein said polypeptide has at least 90%, such as, e.g.at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identitywith SEQ ID NO: 1 is provided. In another aspect, a polypeptide havingat least 96.5% sequence identity to SEQ ID NO: 2 such as wherein saidpolypeptide has at least 97%, such as, e.g. at least 98% or at least 99%sequence identity with SEQ ID NO: 2. In one aspect, the polypeptidesdisclosed herein consist of at the most 975 amino acid residues, suchas, e.g. at most 970 amino acid residues, such as at most 950 amino acidresidues, such as at most 940 amino acid residues, at most 930 aminoacid residues, at most 920 amino acid residues, at most 910 amino acidresidues, at most 900 amino acid residues, at most 895 amino acidresidues or at most 890 amino acid residues, is provided. In one aspect,a particular polypeptide consists of 887 or 965 amino acid residues, isprovided. In one aspect, a polypeptide comprising an amino acid sequencehaving at least 97% sequence identity with SEQ ID NO: 2 such as whereinsaid sequence identity is at least 98%, such as, e.g. at least 99% or atleast 100% sequence identity, wherein said polypeptide consists of atmost 975 amino acid residues, such as, e.g. at most 970 or at least 965amino acid residues, is provided. In one aspect, a polypeptidecomprising an amino acid sequence having at least 97% sequence identitywith SEQ ID NO: 2, wherein said polypeptide consists of at most 975amino acid residues, is provided.

In a further preferred aspect, a polypeptide which comprises SEQ IDNO:1, 2, 3, 4 or 5, is provided. In yet a preferred aspect, apolypeptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3,4, or 5, especially a polypeptide consisting of the amino acid sequenceof SEQ ID NO: 1 or 2, is provided.

In a further aspect, a polypeptide comprising an amino acid sequencehaving at least 96.5% sequence identity with SEQ ID NO: 3 such aswherein said sequence identity is at least 97%, such as, e.g. at least98%, at least 99% or at least 100% sequence identity, wherein saidpolypeptide consists of at most 1300 amino acid residues, is provided.

In a further aspect, a polypeptide wherein said polypeptide has at least98.5%, such as at least 99% or at least 99.5% sequence identity with SEQID NO: 5, is provided. In one aspect, such a polypeptide consists of atmost 1290 amino acid residues, such as, e.g. at most 1280, at most 1270,at most 1260, at most 1250, at most 1240, at most 1230, at most 1220 orat most 1215 amino acid residues, is provided. In a preferred aspect, apolypeptide which consists of 1211 amino acid residues, is provided.

In a further aspect, a polypeptide wherein said polypeptide has at least96% such as at least at least 97%, such as, e.g., at least 98% or atleast 99% sequence identity with SEQ ID NO: 4, is provided. In oneaspect, a polypeptide which consists of at most 1210 amino acidresidues, such as, e.g. at most 1200, at most 1190, at most 1180, atmost 1170, at most 1160, at most 1150 or at most 1145 amino acidresidues, such as 1142 amino acid residues, is provided.

In a further aspect, a polypeptide wherein said polypeptide has at least96.5% such as at least 97%, such as, e.g., at least 98% or at least 99%sequence identity with SEQ ID NO: 3, is provided. In one aspect, apolypeptide which consists of at most 1130 amino acid residues, such as,e.g. at the most 1120, at the most 1110, at the most 1100, at the most1090, at the most 1080, at the most 1070, at the most 1060, at the most1050, at the most 1055 or at the most 1040 amino acid residues, isprovided. In a preferred aspect, a polypeptide which consists of 1038amino acid residues, is provided.

In a further aspect, the polypeptides disclosed herein has a ratio oftransgalactosylation activity above 100% such as above 150%, 175% or200%.

Proteins are generally comprised of one or more functional regions,commonly termed domains. The presence of different domains in varyingcombinations in different proteins gives rise to the diverse repertoireof proteins found in nature. One way of describing the domains are bythe help of the Pfam database which is a large collection of proteindomain families as described in “The Pfam protein families database”: R.D. Finn, J. Mistry, J. Tate, P. Coggill, A. Heger, J. E. Pollington, O.L. Gavin, P. Gunesekaran, G. Ceric, K. Forslund, L. Holm, E. L.Sonnhammer, S. R. Eddy, A. Bateman Nucleic Acids Research (2010)Database Issue 38:D211-222. Each family is represented by multiplesequence alignments and hidden Markov models (HMMs). In a furtheraspect, the present inventors have found that the herein providedpolypeptide(s) contains one or more of the Pfam domains Glyco_hydro2N(PF02837), Glyco_hydro (PF00703), Glyco_hydro 2C (PF02836) and BacterialIg-like domain (group 4) (PF07532). In one aspect, the herein providedpolypeptide(s) contains Glyco_hydro2N (PF02837), Glyco_hydro (PF00703),Glyco_hydro 2C (PF02836) and Bacterial Ig-like domain (group 4)(PF07532).

In one aspect, the polypeptides have useful transgalactosylatingactivity over a range of pH of 4-9, such as 5-8, such as 5.5-7.5, suchas 6.5-7.5.

The present invention encompasses polypeptides having a certain degreeof sequence identity or sequence homology with amino acid sequence(s)defined herein or with a polypeptide having the specific propertiesdefined herein. The present invention encompasses, in particular,peptides having a degree of sequence identity with any one of SEQ ID NO:1, 2, 3, 4 or 5, defined below, or homologues thereof.

In one aspect, the homologous amino acid sequence and/or nucleotidesequence should provide and/or encode a polypeptide which retains thefunctional transgalactosylating activity and/or enhances thetransgalactosylating activity compared to a polypeptide of SEQ ID NO: 1,2, 3, 4 or 5.

In the present context, a homologous sequence is taken to include anamino acid sequence which may be at least 66%, 70%, 75%, 78%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98% or at least 99%, identical to the subject sequence.Typically, the homologues will comprise the same active sites etc. asthe subject amino acid sequence. Although homology can also beconsidered in terms of similarity (i.e. amino acid residues havingsimilar chemical properties/functions), in the context of the presentinvention it is preferred to express homology in terms of sequenceidentity.

Thus, the present invention also encompasses variants, homologues andderivatives of any amino acid sequence of a protein or polypeptide asdefined herein, particularly those of SEQ ID NO: 1, 2, 3, 4 or 5 definedbelow.

The sequences, particularly those of variants, homologues andderivatives of SEQ ID NO: 1, 2, 3, 4 or 5 defined below, may also havedeletions, insertions or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalentsubstance. Deliberate amino acid substitutions may be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe secondary binding activity of the substance is retained. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine,glycine, alanine, asparagine, glutamine, serine, threonine,phenylalanine, and tyrosine.

The present invention also encompasses conservative substitution(substitution and replacement are both used herein to mean theinterchange of an existing amino acid residue, with an alternativeresidue) that may occur i.e. like-for-like substitution such as basicfor basic, acidic for acidic, polar for polar etc. Non-conservativesubstitution may also occur i.e. from one class of residue to another oralternatively involving the inclusion of unnatural amino acids such asornithine (hereinafter referred to as Z), diaminobutyric acid ornithine(hereinafter referred to as B), norleucine ornithine (hereinafterreferred to as O), pyriylalanine, thienylalanine, naphthylalanine andphenylglycine.

Conservative substitutions that may be made are, for example within thegroups of basic amino acids (Arginine, Lysine and Histidine), acidicamino acids (glutamic acid and aspartic acid), aliphatic amino acids(Alanine, Valine, Leucine, Isoleucine), polar amino acids (Glutamine,Asparagine, Serine, Threonine), aromatic amino acids (Phenylalanine,Tryptophan and Tyrosine), hydroxyl amino acids (Serine, Threonine),large amino acids (Phenylalanine and Tryptophan) and small amino acids(Glycine, Alanine).

In one aspect, the polypeptide sequence used in the present invention isin a purified form. In one aspect, the polypeptide or protein for use inthe present invention is in an isolated form.

In one aspect, the polypeptide of the present invention is recombinantlyproduced.

The variant polypeptides include a polypeptide having a certain percent,e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, ofsequence identity with SEQ ID NO: 1 or 2.

The variant polypeptides include a polypeptide having a certain percent,e.g., at least 96%, 97%, 98%, or 99%, of sequence identity with SEQ IDNO: 3, 4 or 5.

In one aspect, the polypeptides disclosed herein comprises an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity to the amino acid sequence of the maturepolypeptide encoded by the nucleotide sequence encoding thetransgalatosylase contained in Bifidobacterium bifidum DSM20215 shownherein as SEQ ID NO: 22. All considerations and limitations relating tosequence identities and functionality discussed in terms of the SEQ IDNO: 1, 2, 3, 4 or 5 apply mutatis mutandis to sequence identities andfunctionality of these polypeptides and nucleotides.

In one aspect, the subject amino acid sequence is SEQ ID NO: 1, 2, 3, 4or 5, and the subject nucleotide sequence preferably is SEQ ID NO: 9,10, 11, 12 or 13.

In one aspect, the polypeptide is a fragment having one or more(several) amino acids deleted from the amino and/or carboxyl terminus ofthe polypeptide of SEQ ID NO: 1, 2, 3, 4 or 5; wherein the fragment hastransgalactosylating activity.

In one aspect, a fragment contains at least 500, 550, 600, 650, 700,750, 800, 850, 900, 950, or 1000 amino acid residues

In a further aspect, the length of the polypeptide variant is 500 to1300 amino acid residues. In a further aspect, the length of thepolypeptide variant is 600 to 1300 amino acids. In a further aspect, thelength of the polypeptide variant is 700 to 1300 amino acids. In afurther aspect, the length of the polypeptide variant is 800 to 1300amino acids. In a further aspect, the length of the polypeptide variantis 800 to 1300 amino acids.

Polypeptide Variants of SEQ ID NO: 1, 2, 3, 4 or 5

In one aspect, a variant of SEQ ID NO: 1, 2, 3, 4 or 5 having asubstitution at one or more positions which effects an altered propertysuch as improved transgalactosylation, relative to SEQ ID NO: 1, 2, 3, 4or 5, is provided. Such variant polypeptides are also referred to inthis document for convenience as “variant polypeptide”, “polypeptidevariant” or “variant”. In one aspect, the polypeptides as defined hereinhave an improved transgalactosylating activity as compared to thepolypeptide of SEQ ID NO: 1, 2, 3, 4 or 5. In another aspect, thepolypeptides as defined herein have an improved reaction velocity ascompared to the polypeptide of SEQ ID NO: 1, 2, 3, 4 or 5.

In one aspect, the polypeptides and variants as defined herein exhibitenzyme activity. In one aspect, the polypeptides and the variantpolypeptides described herein comprise transgalactosylation activity.

In one aspect, the ratio of transgalactosylatingactivity:β-galactosidase activity is at least 0.5, such as at least 1,such as at least 1.5, or such as at least 2 after 30 min. reaction suchas above a concentration of 3% w/w initial lactose concentration.

In one aspect, the ratio of transgalactosylatingactivity:β-galactosidase activity is at least 2.5, such as at least 3,such as at least 4, such as at least 5, such as at least 6, such as atleast 7, such as at least 8, such as at least 9, such as at least 10,such as at least 11, or such as at least 12 after 30 min. reaction suchas above a concentration of 3% w/w initial lactose concentration.

In one aspect, the polypeptides and the variants as defined herein arederivable from microbial sources, in particular from a filamentousfungus or yeast, or from a bacterium. The enzyme may, e.g., be derivedfrom a strain of Agaricus, e.g. A. bisporus; Ascovaginospora;Aspergillus, e.g. A. niger, A. awamori, A. foetidus, A. japonicus, A.oryzae; Candida; Chaetomium; Chaetotomastia; Dictyostelium, e.g. D.discoideum; Kiuveromyces, e.g. K. fragilis, K. lactis; Mucor, e.g. M.javanicus, M. mucedo, M. subtilissimus; Neurospora, e.g. N. crassa;Rhizomucor, e.g. R. pusillus; Rhizopus, e.g. R. arrhizus, R. japonicus,R. stolonifer; Sclerotinia, e.g. S. libertiana; Torula; Torulopsis;Trichophyton, e.g. T. rubrum; Whetzelinia, e.g. W. sclerotiorum;Bacillus, e.g. B. coagulans, B. circulans, B. megaterium, B. novalis, B.subtilis, B. pumilus, B. stearothermophilus, B. thuringiensis;Bifidobacterium, e.g. B. Iongum, B. bifidum, B. animalis;Chryseobacterium; Citrobacter, e.g. C. freundii; Clostridium, e.g. C.perfringens; Diplodia, e.g. D. gossypina; Enterobacter, e.g. E.aerogenes, E. cloacae Edwardsiella, E. tarda; Erwinia, e.g. E.herbicola; Escherichia, e.g. E. coli; Klebsiella, e.g. K. pneumoniae;Miriococcum; Myrothesium; Mucor; Neurospora, e.g. N. crassa; Proteus,e.g. P. vulgaris; Providencia, e.g. P. stuartii; Pycnoporus, e.g.Pycnoporus cinnabarinus, Pycnoporus sanguineus; Ruminococcus, e.g. R.torques; Salmonella, e.g. S. typhimurium; Serratia, e.g. S.liquefasciens, S. marcescens; Shigella, e.g. S. flexneri; Streptomyces,e.g. S. antibioticus, S. castaneoglobisporus, S. violeceoruber;Trametes; Trichoderma, e.g. T. reesei, T. viride; Yersinia, e.g. Y.enterocolitica.

An isolated and/or purified polypeptide comprising a polypeptide or avariant polypeptide as defined herein is provided. In one embodiment,the variant polypeptide is a mature form of the polypeptide (SEQ ID NO:1, 2, 3, 4 or 5). In one aspect, the variants include a C-terminaldomain.

In one aspect, a variant polypeptide as defined herein includes variantswherein between one and about 25 amino acid residues have been added ordeleted with respect to SEQ ID NO: 1, 2, 3, 4 or 5. In one aspect, avariant polypeptide as defined herein includes variants wherein betweenone and 25 amino acid residues have been substituted, added or deletedwith respect to SEQ ID NO: 1, 2, 3, 4 or 5. In one aspect, the varianthas the amino acid sequence of SEQ ID NO: 1, 2, 3, 4 or 5, wherein anynumber between one and about 25 amino acids have been substituted. In afurther aspect, the variant has the amino acid sequence of SEQ ID NO: 1,2, 3, 4 or 5, wherein any number between three and twelve amino acidshas been substituted. In a further aspect, the variant has the aminoacid sequence of SEQ ID NO: 1, 2, 3, 4 or 5, wherein any number betweenfive and nine amino acids has been substituted.

In one aspect, at least two, in another aspect at least three, and yetin another aspect at least five amino acids of SEQ ID NO: 1, 2, 3, 4 or5 have been substituted.

In one aspect, the herein disclosed polypeptide(s) has the sequence of1, 2, 3, 4 or 5.

In one aspect, the herein disclosed polypeptide(s) has the sequence ofSEQ ID NO: 1, 2, 3, 4 or 5, wherein the 10, such as 9, such as 8, suchas 7, such as 6, such 5, such as 4, such as 3, such as 2, such as 1amino acid in the N-terminal end are substituted and/or deleted.

Enzymes and enzyme variants thereof can be characterized by theirnucleic acid and primary polypeptide sequences, by three dimensionalstructural modeling, and/or by their specific activity. Additionalcharacteristics of the polypeptide or polypeptide variants as definedherein include stability, pH range, oxidation stability, andthermostability, for example. Levels of expression and enzyme activitycan be assessed using standard assays known to the artisan skilled inthis field. In another aspect, variants demonstrate improved performancecharacteristics relative to the polypeptide with SEQ ID NO: 1, 2, 3, 4or 5, such as improved stability at high temperatures, e.g., 65-85° C.

A polypeptide variant is provided as defined herein with an amino acidsequence having at least about 66%, 68%, 70%, 72%, 74%, 78%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with thepolypeptide of SEQ ID NO: 1, 2, 3, 4 or 5.

Nucleotides

In one aspect, the present invention relates to isolated polypeptideshaving transgalactosylating activity as stated above which are encodedby polynucleotides which hybridize under very low stringency conditions,preferably low stringency conditions, more preferably medium stringencyconditions, more preferably medium-high stringency conditions, even morepreferably high stringency conditions, and most preferably very highstringency conditions with i) the nucleic acid sequence comprised in SEQID NO: 9, 10, 11, 12 or 13 encoding the mature polypeptide of SEQ ID NO:1, 2, 3, 4 or 5; ii) the cDNA sequence of i) or iii) the complementarystrand of i) or ii), (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,New York). A subsequence of SEQ ID NO: 9, 10, 11, 12 or 13 contains atleast 100 contiguous nucleotides or preferably at least 200 contiguousnucleotides. Moreover, the subsequence may encode a polypeptide fragmentwhich has lactase activity.

The nucleotide sequence of SEQ ID NO: 9, 10, 11, 12 or 13 or asubsequence thereof, as well as the amino acid sequence of SEQ ID NO: 1,2, 3, 4 or 5 or a fragment thereof, may be used to design a nucleic acidprobe to identify and clone DNA encoding polypeptides havingtransgalactosylase activity from strains of different genera or speciesaccording to methods well known in the art. In particular, such probescan be used for hybridization with the genomic or cDNA of the genus orspecies of interest, following standard Southern blotting procedures, inorder to identify and isolate the corresponding gene therein. Suchprobes can be considerably shorter than the entire sequence, but shouldbe at least 14, preferably at least 25, more preferably at least 35, andmost preferably at least 70 nucleotides in length. It is, however,preferred that the nucleic acid probe is at least 100 nucleotides inlength. For example, the nucleic acid probe may be at least 200nucleotides, preferably at least 300 nucleotides, more preferably atleast 400 nucleotides, or most preferably at least 500 nucleotides inlength. Even longer probes may be used, e.g., nucleic acid probes whichare at least 600 nucleotides, at least preferably at least 700nucleotides, more preferably at least 800 nucleotides, or mostpreferably at least 900 nucleotides in length. Both DNA and RNA probescan be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

A genomic DNA library prepared from such other organisms may, therefore,be screened for DNA which hybridizes with the probes described above andwhich encodes a polypeptide having lactase activity. Genomic or otherDNA from such other organisms may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA which is homologous with SEQ ID NO: 9,10, 11, 12 or 13 or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labelled nucleic acid probecorresponding to the nucleotide sequence shown in SEQ ID NO: 9, 10, 11,12 or 13, its complementary strand, or a subsequence thereof, under verylow to very high stringency conditions. Molecules to which the nucleicacid probe hybridizes under these conditions can be detected using X-rayfilm.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding region of SEQ ID NO: 9, 10, 11, 12 or 13.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 g/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

In a particular embodiment, the wash is conducted using 0.2×SSC, 0.2%SDS preferably at least at 45° C. (very low stringency), more preferablyat least at 50° C. (low stringency), more preferably at least at 55° C.(medium stringency), more preferably at least at 60° C. (medium-highstringency), even more preferably at least at 65° C. (high stringency),and most preferably at least at 70° C. (very high stringency). Inanother particular embodiment, the wash is conducted using 0.1×SSC, 0.2%SDS preferably at least at 45° C. (very low stringency), more preferablyat least at 50° C. (low stringency), more preferably at least at 55° C.(medium stringency), more preferably at least at 60° C. (medium-highstringency), even more preferably at least at 65° C. (high stringency),and most preferably at least at 70° C. (very high stringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

Under salt-containing hybridization conditions, the effective T_(m) iswhat controls the degree of identity required between the probe and thefilter bound DNA for successful hybridization. The effective T_(m) maybe determined using the formula below to determine the degree ofidentity required for two DNAs to hybridize under various stringencyconditions.

Effective T _(m)=81.5+16.6(log M[Na⁺])+0.41(% G+C)−0.72(% formamide)

(See www.ndsu.nodak.edu/instruct/mcclean/plsc731/dna/dna6.htm)

The G+C content of SEQ ID NO: 10 is 42% and the G+C content of SEQ IDNO: 11 is 44%. For medium stringency, the formamide is 35% and the Na⁺concentration for 5×SSPE is 0.75 M.

Another relevant relationship is that a 1% mismatch of two DNAs lowersthe T_(m) by 1.4° C. To determine the degree of identity required fortwo DNAs to hybridize under medium stringency conditions at 42° C., thefollowing formula is used:

% Homology=100−[(Effective T _(m)−Hybridization Temperature)/1.4]

(See www.ndsu.nodak.edu/instruct/mcclean/plsc731/dna/dna6.htm)

The variant nucleic acids include a polynucleotide having a certainpercent, e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, ofsequence identity with the nucleic acid encoding SEQ ID NO: 1, 2, 3, 4or 5. In one aspect, a nucleic acid capable of encoding a polypeptide asdisclosed herein, is provided. In a further aspect, the herein disclosednucleic acid has a nucleic acid sequence which is at least 60%, such asat least 65%, such as at least 70%, such as at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 99% identical SEQ ID NO: 9, 10, 11, 12 or 13.

In one aspect, a plasmid comprising a nucleic acid as described herein,is provided.

In one aspect, an expression vector comprising a nucleic acid asdescribed herein, or capable of expressing a polypeptide as describedherein, is provided.

A nucleic acid complementary to a nucleic acid encoding any of thepolypeptide variants as defined herein set forth herein is provided.Additionally, a nucleic acid capable of hybridizing to the complement isprovided. In another embodiment, the sequence for use in the methods andcompositions described here is a synthetic sequence. It includes, but isnot limited to, sequences made with optimal codon usage for expressionin host organisms, such as yeast. The polypeptide variants as providedherein may be produced synthetically or through recombinant expressionin a host cell, according to procedures well known in the art. In oneaspect, the herein disclosed polypeptide(s) is recombinantpolypeptide(s). The expressed polypeptide variant as defined hereinoptionally is isolated prior to use.

In another embodiment, the polypeptide variant as defined herein ispurified following expression. Methods of genetic modification andrecombinant production of polypeptide variants are described, forexample, in U.S. Pat. Nos. 7,371,552, 7,166,453; 6,890,572; and6,667,065; and U.S. Published Application Nos. 2007/0141693;2007/0072270; 2007/0020731; 2007/0020727; 2006/0073583; 2006/0019347;2006/0018997; 2006/0008890; 2006/0008888; and 2005/0137111. The relevantteachings of these disclosures, including polypeptide-encodingpolynucleotide sequences, primers, vectors, selection methods, hostcells, purification and reconstitution of expressed polypeptidevariants, and characterization of polypeptide variants as definedherein, including useful buffers, pH ranges, Ca²⁺ concentrations,substrate concentrations and enzyme concentrations for enzymatic assays,are herein incorporated by reference.

A nucleic acid sequence is provided encoding the protein of SEQ ID NO:1, 2, 3, 4 or 5 or a nucleic acid sequence having at least about 66%,68%, 70%, 72%, 74%, 78%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity with a nucleic acid encoding the protein of SEQ ID NO:1, 2, 3, 4 or 5. In one embodiment, the nucleic acid sequence has atleast about 60%, 66%, 68%, 70%, 72%, 74%, 78%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% sequence identity to the nucleic acid of SEQ ID NO: 9,10, 11, 12 or 13.

Vectors

In one aspect, the invention relates to a vector comprising apolynucleotide. In one aspect, a bacterial cell comprises the vector. Insome embodiments, a DNA construct comprising a nucleic acid encoding avariant is transferred to a host cell in an expression vector thatcomprises regulatory sequences operably linked to an encoding sequence.The vector may be any vector that can be integrated into a fungal hostcell genome and replicated when introduced into the host cell. The FGSCCatalogue of Strains, University of Missouri, lists suitable vectors.Additional examples of suitable expression and/or integration vectorsare provided in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL,3^(rd) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (2001); Bennett et al., MORE GENE MANIPULATIONS IN FUNGI, AcademicPress, San Diego (1991), pp. 396-428; and U.S. Pat. No. 5,874,276.Exemplary vectors include pFB6, pBR322, PUC18, pUC100 and pENTR/D,pDON™201, pDONR™221, pENTR™, pGEM®3Z and pGEM®4Z. Exemplary for use inbacterial cells include pBR322 and pUC19, which permit replication in E.coli, and pE194, for example, which permits replication in Bacillus.

In some embodiments, a nucleic acid encoding a variant is operablylinked to a suitable promoter, which allows transcription in the hostcell. The promoter may be derived from genes encoding proteins eitherhomologous or heterologous to the host cell. Suitable non-limitingexamples of promoters include cbh1, cbh2, egl1, and egl2 promoters. Inone embodiment, the promoter is one that is native to the host cell. Forexample, when P. saccharophila is the host, the promoter is a native P.saccharophila promoter. An “inducible promoter” is a promoter that isactive under environmental or developmental regulation. In anotherembodiment, the promoter is one that is heterologous to the host cell.

In some embodiments, the coding sequence is operably linked to a DNAsequence encoding a signal sequence. In another aspect, a representativesignal peptide is SEQ ID NO: 27. A representative signal peptide is SEQID NO: 9 which is the native signal sequence of the Bacillus subtilisaprE precursor. In other embodiments, the DNA encoding the signalsequence is replaced with a nucleotide sequence encoding a signalsequence from other extra-cellular Bacillus subtilis pre-cursors. In oneembodiment, the polynucleotide that encodes the signal sequence isimmediately upstream and in-frame of the polynucleotide that encodes thepolypeptide. The signal sequence may be selected from the same speciesas the host cell.

In additional embodiments, a signal sequence and a promoter sequencecomprising a DNA construct or vector to be introduced into a fungal hostcell are derived from the same source. In some embodiments, theexpression vector also includes a termination sequence. In oneembodiment, the termination sequence and the promoter sequence arederived from the same source. In another embodiment, the terminationsequence is homologous to the host cell.

In some embodiments, an expression vector includes a selectable marker.Examples of suitable selectable markers include those that conferresistance to antimicrobial agents, e.g., hygromycin or phleomycin.Nutritional selective markers also are suitable and include amdS, argB,and pyr4. In one embodiment, the selective marker is the amdS gene,which encodes the enzyme acetamidase; it allows transformed cells togrow on acetamide as a nitrogen source. The use of an A. nidulans amdSgene as a selective marker is described in Kelley et al., EMBO J. 4:475-479 (1985) and Penttila et al., Gene 61: 155-164 (1987).

A suitable expression vector comprising a DNA construct with apolynucleotide encoding a variant may be any vector that is capable ofreplicating autonomously in a given host organism or integrating intothe DNA of the host. In some embodiments, the expression vector is aplasmid. In some embodiments, two types of expression vectors forobtaining expression of genes are contemplated. The first expressionvector comprises DNA sequences in which the promoter, coding region, andterminator all originate from the gene to be expressed. In someembodiments, gene truncation is obtained by deleting undesired DNAsequences to leave the domain to be expressed under control of its owntranscriptional and translational regulatory sequences. The second typeof expression vector is preassembled and contains sequences required forhigh-level transcription and a selectable marker. In some embodiments,the coding region for a gene or part thereof is inserted into thisgeneral-purpose expression vector, such that it is under thetranscriptional control of the expression construct promoter andterminator sequences. In some embodiments, genes or part thereof areinserted downstream of the strong cbh1 promoter.

Expression Hosts/Host Cells

In a further aspect, a host cell comprising, preferably transformedwith, a plasmid as described herein or an expression vector as describedherein, is provided.

In a further aspect, a cell capable of expressing a polypeptide asdescribed herein, is provided.

In one aspect, the host cell as described herein, or the cell asdescribed herein is a bacterial, fungal or yeast cell.

In a further aspect, the host cell is selected from the group consistingof Ruminococcus, Bifidobacterium, Lactococcus, Lactobacillus,Streptococcus, Leuconostoc, Escherichia, Bacillus, Streptomyces,Saccharomyces, Kluyveromyces, Candida, Torula, Torulopsís andAspergillus.

In a further aspect, the host cell is selected from the group consistingof Ruminococcus hansenii, Bifidobacterium breve, Bifidobacterium longum,Bifidobacterium infantis, Bifidobacterium bifidum and Lactococcuslactis.

In another embodiment, suitable host cells include a Gram positivebacterium selected from the group consisting of Bacillus subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus, B. thuringiensis, Streptomyces lividans, or S. murinus; or aGram negative bacterium, wherein said Gram negative bacterium isEscherichia coli or a Pseudomonas species. In one aspect, the host cellis a B. subtilis or B. licheniformis. In one embodiment, the host cellis B. subtilis, and the expressed protein is engineered to comprise a B.subtilis signal sequence, as set forth in further detail below. In oneaspect, the host cell expresses the polynucleotide as set out in theclaims.

In some embodiments, a host cell is genetically engineered to express apolypeptide variant as defined herein with an amino acid sequence havingat least about 66%, 68%, 70%, 72%, 74%, 78%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the polypeptide ofSEQ ID NO: 1, 2, 3, 4 or 5. In some embodiments, the polynucleotideencoding a polypeptide variant as defined herein will have a nucleicacid sequence encoding the protein of SEQ ID NO: 1, 2, 3, 4 or 5 or anucleic acid sequence having at least about 66%, 68%, 70%, 72%, 74%,78%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identitywith a nucleic acid encoding the protein of SEQ ID NO: 1, 2, 3, 4 or 5.In one embodiment, the nucleic acid sequence has at least about 60%,66%, 68%, 70%, 72%, 74%, 78%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%sequence identity to the nucleic acid of SEQ ID NO: 9, 10, 11, 12 or 13.

Methods for Producing Polypeptides

In a further aspect, a method of expressing a polypeptide as describedherein comprises obtaining a host cell or a cell as described herein andexpressing the polypeptide from the cell or host cell, and optionallypurifying the polypeptide.

An expression characteristic means an altered level of expression of thevariant, when the variant is produced in a particular host cell.Expression generally relates to the amount of active variant that isrecoverable from a fermentation broth using standard techniques known inthis art over a given amount of time. Expression also can relate to theamount or rate of variant produced within the host cell or secreted bythe host cell. Expression also can relate to the rate of translation ofthe mRNA encoding the variant polypeptide.

Transformation, Expression and Culture of Host Cells

Introduction of a DNA construct or vector into a host cell includestechniques such as transformation; electroporation; nuclearmicroinjection; transduction; transfection, e.g., lipofection mediatedand DEAE-Dextrin mediated transfection; incubation with calciumphosphate DNA precipitate; high velocity bombardment with DNA-coatedmicroprojectiles; and protoplast fusion. General transformationtechniques are known in the art. See, e.g., Ausubel et al. (1987),supra, chapter 9; Sambrook et al. (2001), supra; and Campbell et al.,Curr. Genet. 16: 53-56 (1989). The expression of heterologous protein inTrichoderma is described, for example, in U.S. Pat. No. 6,022,725; U.S.Pat. No. 6,268,328; Harkki et al., Enzyme Microb. Technol. 13: 227-233(1991); Harkki et al., BioTechnol. 7: 596-603 (1989); EP 244,234; and EP215,594. In one embodiment, genetically stable transformants areconstructed with vector systems whereby the nucleic acid encoding avariant is stably integrated into a host cell chromosome. Transformantsare then purified by known techniques.

In one non-limiting example, stable transformants including an amdSmarker are distinguished from unstable transformants by their fastergrowth rate and the formation of circular colonies with a smooth, ratherthan ragged outline on solid culture medium containing acetamide.Additionally, in some cases a further test of stability is conducted bygrowing the transformants on solid non-selective medium, e.g., a mediumthat lacks acetamide, harvesting spores from this culture medium anddetermining the percentage of these spores that subsequently germinateand grow on selective medium containing acetamide. Other methods knownin the art may be used to select transformants.

Identification of Activity

To evaluate the expression of a variant in a host cell, assays canmeasure the expressed protein, corresponding mRNA, or β-galactosidaseactivity. For example, suitable assays include Northern and Southernblotting, RT-PCR (reverse transcriptase polymerase chain reaction), andin situ hybridization, using an appropriately labeled hybridizing probe.Suitable assays also include measuring activity in a sample. Suitableassays of the activity of the variant include, but are not limited to,ONPG based assays or determining glucose in reaction mixtures such forexample described in the methods and examples herein.

Methods for Purifying Herein Disclosed Polypeptides

In general, a variant produced in cell culture is secreted into themedium and may be purified or isolated, e.g., by removing unwantedcomponents from the cell culture medium. In some cases, a variant may berecovered from a cell lysate. In such cases, the enzyme is purified fromthe cells in which it was produced using techniques routinely employedby those of skill in the art. Examples include, but are not limited to,affinity chromatography, ion-exchange chromatographic methods, includinghigh resolution ion-exchange, hydrophobic interaction chromatography,two-phase partitioning, ethanol precipitation, reverse phase HPLC,chromatography on silica or on a cation-exchange resin, such as DEAE,chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gelfiltration using Sephadex G-75, for example. Depending on the intendeduse the herein disclosed polypeptide(s) may for example be eitherfreeze-dried or prepared in a solution. In one aspect, the hereindisclosed polypeptide(s) is freeze-dried form. In another aspect, theherein disclosed polypeptide(s) is in solution.

Compositions, Application and Use Methods for Immobilising andFormulation of the Herein Disclosed Polypeptides

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art. Examples are given below of preferred uses of the polypeptidesor polypeptide compositions of the invention.

In one aspect, disclosed herein is a method for producing a food productby treating a substrate comprising lactose with a polypeptide asdescribed herein.

In one aspect, disclosed herein is a method for producing a dairyproduct by treating a milk-based substrate comprising lactose with apolypeptide as described herein.

In one aspect, the substrate comprising lactose is further treated witha hydrolysing beta-galactosidase.

The enzyme preparation, such as in the form of a food ingredientprepared according to the present invention, may be in the form of asolution or as a solid—depending on the use and/or the mode ofapplication and/or the mode of administration. The solid form can beeither as a dried enzyme powder or as a granulated enzyme.

Examples of dry enzyme formulations include spray dried products, mixergranulation products, layered products suc as fluid bed granules,extruded or pelletized granules prilled products, lyophilyzed products.

The enzyme preparation, such as in the form of a food ingredientprepared according to the present invention, may be in the form of asolution or as a solid—depending on the use and/or the mode ofapplication and/or the mode of administration. The solid form can beeither as a dried enzyme powder or as a granulated enzyme.

In one aspect, a composition preferably a food composition, morepreferably a dairy product comprising a cell or a polypeptide asdescribed herein, is provided.

Furthermore, disclosed herein is a composition comprising at least 5%,such as e.g. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% w/w of one ormore polypeptide(s) as disclosed herein based on the total amount ofpolypeptides in the composition having at least 70%,e.g. such as 72%,74%, 74%, 78%, 80%, 82%, 84%, 86%, 88%, 90% sequence identity with SEQID NO: 22. This may be evaluated by using the following techniques knowto a person skilled in the art. The samples to be evaluated aresubjected to SDS-PAGE and visualized using a dye appropriate for proteinquantification, such as for example the Bio-Rad Criterion system. Thegel is then scanned using appropriate densiometic scanner such as forexample the Bio-Rad Criterion system and the resulting picture isensured to be in the dynamic range. The bands corresponding to anyvariant/fragment derived from SEQ ID NO: 8 are quantified and thepercentage of the polypeptides are calculated as: Percentage ofpolypeptide in question=polypeptide in question/(sum of all polypeptidesexhibiting transgalactosylating activity)*100. The total number ofpolypeptides variants/fragments derived from SEQ ID NO:8 in thecomposition can be determined by detecting fragment derived from SEQ IDNO:8 by western blotting using a polyclonal antibody by methods know toa person skilled in the art.

In one aspect, the composition according to the present inventioncomprises one or more polypeptide(s) selected from the group consistingof a polypeptide consisting of SEQ ID NO: 1, 2, 3, 4 and 5. In a furtheraspect, the composition comprises one or more polypeptide(s) selectedfrom the group consisting of a polypeptide consisting of SEQ ID NO: 1, 2and 3. In yet a further aspect, the composition comprises one or morepolypeptide(s) selected from the group consisting of a polypeptideconsisting of SEQ ID NO: 1 and 2.

In one aspect the invention provides an enzyme complex preparationcomprising the enzyme complex according to the invention, an enzymecarrier and optionally a stabilizer and/or a preservative.

In yet a further aspect of the invention, the enzyme carrier is selectedfrom the group consisting of glycerol or water.

In a further aspect, the preparation/composition comprises a stabilizer.In one aspect, the stabilizer is selected from the group consisting ofinorganic salts, polyols, sugars and combinations thereof. In oneaspect, the stabilizer is an inorganic salt such as potassium chloride.In another aspect, the polyol is glycerol, propylene glycol, orsorbitol. In yet another aspect, the sugar is a small-moleculecarbohydrate, in particular any of several sweet-tasting ones such asglucose, galactose, fructose and saccharose.

In yet at further aspect, the preparation comprises a preservative. Inone aspect, the preservative is methyl paraben, propyl paraben,benzoate, sorbate or other food approved preservatives or a mixturethereof.

The method of the invention can be practiced with immobilized enzymes,e.g. an immobilized lactase or other galactooligosaccharide producingenzymes. The enzyme can be immobilized on any organic or inorganicsupport. Exemplary inorganic supports include alumina, celite,Dowex-1-chloride, glass beads and silica gel. Exemplary organic supportsinclude DEAE-cellulose, alginate hydrogels or alginate beads orequivalents. In various aspects of the invention, immobilization of thelactase can be optimized by physical adsorption on to the inorganicsupport. Enzymes used to practice the invention can be immobilized indifferent media, including water, Tris-HCl buffer and phosphate bufferedsolution. The enzyme can be immobilized to any type of substrate, e.g.filters, fibers, columns, beads, colloids, gels, hydrogels, meshes andthe like.

In one aspect, a method for producing a dairy product by treating amilk-based substrate comprising lactose with a polypeptide as describedherein is provided. In a further aspect, a method for producing a dairyproduct by treating a milk-based substrate comprising lactose with apolypeptide having a relative transgalactosylation activity above 60%,such as above 70%, such as above 75% after 15 min. reaction, isprovided. In one aspect, the relative transgalactosylation activity isabove 3 after 30 min. reaction. In a further aspect, the relativetransgalactosylation activity is above 6 after 30 min. reaction. In yeta further aspect, the relative transgalactosylation activity is above 12after 30 min. reaction. In one aspect, a method is provided, wherein thetreatment with a polypeptide as described herein takes place at anoptimal temperature for the activity of the enzyme. In a further aspect,the polypeptide is added to the milk-based substrate at a concentrationof 0.01-1000 ppm. In yet a further aspect, the polypeptide is added tothe milk-based substrate at a concentration of 0.1-100 ppm. In a furtheraspect, the polypeptide is added to the milk-based substrate at aconcentration of 1-10 ppm. In one aspect, a method further comprisingfermenting a substrate such as a dairy product with a microorganism, isprovided. In a further aspect, the dairy product is yogurt. In a furtheraspect, the treatment with the polypeptide and the microorganism isperformed essentially at the same time. In one aspect, the polypeptideand the microorganism are added to the milk-based substrate essentiallyat the same time. In one aspect, a dairy product comprising a cell or apolypeptide as described herein, is provided. In one aspect, thepolypeptide as defined herein is added in a concentration of 0.01-1000ppm. In one aspect, a dairy product comprising an inactivatedpolypeptide as defined herein, is provided. In one aspect, a dairyproduct comprising an inactivated polypeptide as defined herein in aconcentration of 0.01-1000 ppm, is provided. In one aspect, a dairyproduct comprising GOS formed in situ by a polypeptide as definedherein, is provided. In one aspect, a dairy product comprising a cell asdefined herein, is provided.

A dairy product as described herein may be, e.g., skim milk, low fatmilk, whole milk, cream, UHT milk, milk having an extended shelf life, afermented milk product, cheese, yoghurt, butter, dairy spread, buttermilk, acidified milk drink, sour cream, whey based drink, ice cream,condensed milk, dulce de leche or a flavoured milk drink. A dairyproduct may be manufactured by any method known in the art.

A dairy product may additionally comprise non-milk components, e.g.vegetable components such as, e.g., vegetable oil, vegetable protein,and/or vegetable carbohydrates. Dairy products may also comprise furtheradditives such as, e.g., enzymes, flavouring agents, microbial culturessuch as probiotic cultures, salts, sweeteners, sugars, acids, fruit,fruit juices, or any other component known in the art as a component of,or additive to, a dairy product.

In one embodiment of the invention, one or more milk components and/ormilk fractions account for at least 50% (weight/weight), such as atleast 70%, e.g. at least 80%, preferably at least 90%, of the dairyproduct.

In one embodiment of the invention, one or more milk-based substrateshaving been treated with an enzyme as defined herein havingtransgalactosylating activity account for at least 50% (weight/weight),such as at least 70%, e.g. at least 80%, preferably at least 90%, of thedairy product.

In one embodiment of the invention, the dairy product is a dairy productwhich is not enriched by addition of pre-producedgalacto-oligosaccharides.

In one embodiment of the invention, the polypeptide-treated milk-basedsubstrate is not dried before being used as an ingredient in the dairyproduct.

In one embodiment of the invention, the dairy product is ice cream. Inthe present context, ice cream may be any kind of ice cream such as fullfat ice cream, low fat ice cream, or ice cream based on yoghurt or otherfermented milk products. Ice cream may be manufactured by any methodknown in the art.

In one embodiment of the invention, the dairy product is milk orcondensed milk.

In one embodiment of the invention, the dairy product is UHT milk. UHTmilk in the context of the present invention is milk which has beensubjected to a sterilization procedure which is intended to kill allmicroorganisms, including the bacterial spores. UHT (ultra hightemperature) treatment may be, e.g., heat treatment for 30 seconds at130° C., or heat treatment for one second at 145° C.

In one preferred embodiment of the invention, the dairy product is ESLmilk. ESL milk in the present context is milk which has an extendedshelf life due to microfiltration and/or heat treatment and which isable to stay fresh for at least 15 days, preferably for at least 20days, on the store shelf at 2-5° C.

In another preferred embodiment of the invention, the dairy product is afermented dairy product, e.g., yoghurt.

The microorganisms used for most fermented milk products are selectedfrom the group of bacteria generally referred to as lactic acidbacteria. As used herein, the term “lactic acid bacterium” designates agram-positive, microaerophilic or anaerobic bacterium, which fermentssugars with the production of acids including lactic acid as thepredominantly produced acid, acetic acid and propionic acid. Theindustrially most useful lactic acid bacteria are found within the order“Lactobacillales” which includes Lactococcus spp., Streptococcus spp.,Lactobacillus spp., Leuconostoc spp., Pseudoleuconostoc spp.,Pediococcus spp., Brevibacterium spp., Enterococcus spp. andPropionibacterium spp. Additionally, lactic acid producing bacteriabelonging to the group of anaerobic bacteria, bifidobacteria, i.e.Bifidobacterium spp., which are frequently used as food cultures aloneor in combination with lactic acid bacteria, are generally included inthe group of lactic acid bacteria.

Lactic acid bacteria are normally supplied to the dairy industry eitheras frozen or freeze-dried cultures for bulk starter propagation or asso-called “Direct Vat Set” (DVS) cultures, intended for directinoculation into a fermentation vessel or vat for the production of afermented dairy product. Such cultures are in general referred to as“starter cultures” or “starters”.

Commonly used starter culture strains of lactic acid bacteria aregenerally divided into mesophilic organisms having optimum growthtemperatures at about 30° C. and thermophilic organisms having optimumgrowth temperatures in the range of about 40 to about 45° C. Typicalorganisms belonging to the mesophilic group include Lactococcus lactis,Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp.cremoris, Pseudoleuconostoc mesenteroides subsp. cremoris, Pediococcuspentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis,Lactobacillus casei subsp. casei and Lactobacillus paracasei subsp.paracasei. Thermophilic lactic acid bacterial species include asexamples Streptococcus thermophilus, Enterococcus faecium, Lactobacillusdelbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillusdelbrueckii subsp. bulgaricus and Lactobacillus acidophilus. Also theanaerobic bacteria belonging to the genus Bifidobacterium includingBifidobacterium bifidum, Bifidobacterium animalis and Bifidobacteriumlongum are commonly used as dairy starter cultures and are generallyincluded in the group of lactic acid bacteria. Additionally, species ofPropionibacteria are used as dairy starter cultures, in particular inthe manufacture of cheese. Additionally, organisms belonging to theBrevibacterium genus are commonly used as food starter cultures.

Another group of microbial starter cultures are fungal cultures,including yeast cultures and cultures of filamentous fungi, which areparticularly used in the manufacture of certain types of cheese andbeverage. Examples of fungi include Penicillium roqueforti, Penicilliumcandidum, Geotrichum candidum, Torula kefir, Saccharomyces kefir andSaccharomyces cerevisiae.

In one embodiment of the present invention, the microorganism used forfermentation of the milk-based substrate is Lactobacillus casei or amixture of Streptococcus thermophilus and Lactobacillus delbrueckiisubsp. bulgaricus.

Fermentation processes to be used in a method of the present inventionare well known and the person of skill in the art will know how toselect suitable process conditions, such as temperature, oxygen, amountand characteristics of microorganism/s, additives such as e.g.carbohydrates, flavours, minerals, enzymes, and process time. Obviously,fermentation conditions are selected so as to support the achievement ofthe present invention.

As a result of fermentation, pH of the milk-based substrate will belowered. The pH of a fermented dairy product of the invention may be,e.g., in the range 3.5-6, such as in the range 3.5-5, preferably in therange 3.8-4.8.

In one aspect, a method of using the polypeptides or using any one ormore of the above mentioned cell types for producing oligosaccharides,is provided. The oligosaccharides comprise, but are not limited tofructooligo-saccharides, galacto-oligosaccharides,isomalto-oligosaccharides, malto-oligosaccharides, lactosucrose andxylo-oligosaccharides.

In one embodiment of the invention, the oligosaccharides are produced byincubating the cell expressing the polypeptide in a medium thatcomprises a disaccharide substrate such as for example lactulose,trehalose, rhamnose, maltose, sucrose, lactose, or cellobiose. Theincubation is carried out under conditions where oligosaccarides areproduced. The cells may be part of a product selected from the groupconsisting of yoghurt, cheese, fermented milk products, dietarysupplements, and probiotic comestible products. Alternatively, theoligosaccharides can be recovered and subsequently be added to theproduct of interest before or after its preparation.

In one aspect, the use of a herein disclosed cell for producing aproduct selected from the group consisting of yoghurt, cheese, fermentedmilk product, dietary supplement and probiotic comestible product, isprovided.

In one aspect, the polypeptides described herein may be used to preparecheese products and in methods for making the cheese products. Cheeseproducts may e.g. be selected from the group consisting of cream cheese,cottage cheese, and process cheese. By adding polypeptides the cheesesmay contain significantly increased levels of galacto-oligosaccharidesand reduced levels of lactose. In one aspect, the lactose levels in thefinal cheese product may be reduced by at least about 25 percent,preferably at least about 50 percent, and more preferably at least about75 percent. The polypeptides may be used to reduce lactose in cheeseproducts to less than about 1 gram per serving, an amount that can betolerated by most lactose-intolerant individuals.

The cheese products provided herein are nutritionally-enhanced cheeseproducts having increased soluble fiber content, reduced caloriccontent, excellent organoleptic properties, improved texture, andflavor. Further, the polypeptides described herein may reduce theglycemic index of the cheese products because GOS are more slowlyabsorbed than lactose or its hydrolysis products. Finally, thepolypeptides may reduce the cost of production of cheese products,particularly cream cheese products, because GOS surprisingly provideimproved texture to the cream cheese product, thus permitting reduceduse of stabilizers, or by allowing for increased moisture contentwithout syneresis.

In a further aspect, a composition comprising a polypeptide as describedherein and a carbohydrate substrate, is provided. In a further aspect,the carbohydrate substrate is a disaccharide. In a further aspect, thedisaccharide is for example lactulose, trehalose, rhamnose, maltose,sucrose, lactose or cellobiose. In yet a further aspect, thecarbohydrate substrate is lactose. The composition is prepared such thatoligosaccarides are produced. The polypeptide as described herein may bepart of a product selected from the group consisting of yoghurt, cheese,fermented milk products, dietary supplements, and probiotic comestibleproducts. In one aspect, a composition comprising a polypeptide asdescribed herein and a stabilizer, is provided. Examples of stabilizersis e.g., a polyol such as, e.g., glycerol or propylene glycol, a sugaror a sugar alcohol, lactic acid, boric acid, or a boric acid derivative(e.g., an aromatic borate ester).

In one aspect, the use of a transgalactosylating polypeptide asdisclosed herein or a cell as disclosed herein, for producinggalacto-oligosaccharides, is provided. In one aspect, the use of atransgalactosylating polypeptide as disclosed herein or a cell asdisclosed herein, for producing galacto-oligosaccharides to be part of aproduct selected from the group consisting of yoghurt, cheese, fermenteddairy products, dietary supplements and probiotic comestible products,is provided. In one aspect, the product is yoghurt, cheese, or fermenteddairy products. In one aspect, the use of a transgalactosylatingpolypeptide as disclosed herein or a cell as disclosed herein, forproducing galacto-oligosaccharides to enhance the growth ofBifidobacterium, is provided. In one aspect, the use of atransgalactosylating polypeptide as disclosed herein or a cell asdisclosed herein, for producing galacto-oligosaccharides to enhance thegrowth of Bifidobacterium in a mixed culture fermentation, is provided.

In one aspect, a process for producing a transgalactosylatingpolypeptide as disclosed herein, comprising culturing a cell asdisclosed herein in a suitable culture medium under conditionspermitting expression of said polypeptide, and recovering the resultingpolypeptide from the culture, is provided. A process for producinggalacto-oligosaccharides, comprising contacting of an polypeptide of asdisclosed herein or a cell as disclosed herein with a milk-basedsolution comprising lactose, is provided.

Addition of oligosaccharides may enhance growth of eitherBifidobacterium alone or of Bifidobacterium in a mixed culture.

The treatment of milk products with enzymes that converts lactose intomonosaccharides or GOS have several advantages. First the products canbe consumed by people with lactose intolerance that would otherwiseexhibit symptoms such as flatulence and diarrhea. Secondly, dairyproducts treated with lactase will have a higher sweetness than similaruntreated products due to the higher perceived sweetness of glucose andgalactose compared to lactose. This effect is particularly interestingfor applications such as yoghurt and ice-cream where high sweetness ofthe end product is desired and this allows for a net reduction ofcarbohydrates in the consumed product. Thirdly, in ice-cream productiona phenomenon termed sandiness is often seen, where the lactose moleculescrystallizes due to the relative low solubility of the lactose. Whenlactose is converted into monosaccharides or GOS the mouth feeling ofthe ice-cream is much improved over the non-treated products. Thepresence of a sandy feeling due to lactose crystallization can beeliminated and the raw material costs can be decreased by replacement ofskimmed milk powder by whey powder. The main effects of the enzymatictreatment were increased sweetness.

In one aspect, the transgalactosylating polypeptide(s) as disclosedherein may be used together with other enzymes such as proteases such aschymosin or rennin, lipases such as phospholipases, amylases,transferases, and lactases. In one aspect, the transgalactosylatingpolypeptide(s) as disclosed herein may be used together with lactase.This may especially be useful when there is a desire to reduce residuallactose after treatment with the transgalactosylating polypeptide(s) asdisclosed herein especially at low lactose levels. A lactase in thecontext of the present invention is any glycoside hydrolase having theability to hydrolyse the disaccharide lactose into constituent galactoseand glucose monomers. The group of lactases comprises but is not limitedto enzymes assigned to subclass EC 3.2.1.108. Enzymes assigned to othersubclasses, such as, e.g., EC 3.2.1.23, may also be lactases in thecontext of the present invention. A lactase in the context of theinvention may have other activities than the lactose hydrolysingactivity, such as for example a transgalactosylating activity. In thecontext of the invention, the lactose hydrolysing activity of thelactase may be referred to as its lactase activity or itsbeta-galactosidase activity. Enzymes having lactase activity to be usedin a method of the present invention may be of animal, of plant or ofmicrobial origin. Preferred enzymes are obtained from microbial sources,in particular from a filamentous fungus or yeast, or from a bacterium.The enzyme may, e.g., be derived from a strain of Agaricus, e.g. A.bisporus; Ascovaginospora; Aspergillus, e.g. A. niger, A. awamori, A.foetidus, A. japonicus, A. oryzae; Candida; Chaetomium; Chaetotomastia;Dictyostelium, e.g. D. discoideum; Kluveromyces, e.g. K. fragilis, K.lactis; Mucor, e.g. M. javanicus, M. mucedo, M. subtilissimus;Neurospora, e.g. N. crassa; Rhizomucor, e.g. R. pusillus; Rhizopus, e.g.R. arrhizus, R. japonicus, R. stolonifer; Sclerotinia, e.g. S.libertiana; Torula; Torulopsis; Trichophyton, e.g. T. rubrum;Whetzelinia, e.g. W. sclerotiorum; Bacillus, e.g. B. coagulans, B.circulans, B. megaterium, B. novalis, B. subtilis, B. pumilus, B.stearothermophilus, B. thuringiensis; Bifidobacterium, e.g. B. longum,B. bifidum, B. animalis; Chryseobacterium; Citrobacter, e.g. C.freundii; Clostridium, e.g. C. perfringens; Diplodia, e.g. D. gossypina;Enterobacter, e.g. E. aerogenes, E. cloacae Edwardsiella, E. tarda;Erwinia, e.g. E. herbicola; Escherichia, e.g. E. coli; Klebsiella, e.g.K. pneumoniae; Miriococcum; Myrothesium; Mucor; Neurospora, e.g. N.crassa; Proteus, e.g. P. vulgaris; Providencia, e.g. P. stuartii;Pycnoporus, e.g. Pycnoporus cinnabarinus, Pycnoporus sanguineus;Ruminococcus, e.g. R. torques; Salmonella, e.g. S. typhimurium;Serratia, e.g. S. liquefasciens, S. marcescens; Shigella, e.g. S.flexneri; Streptomyces, e.g. S. antibioticus, S. castaneoglobisporus, S.violeceoruber; Trametes; Trichoderma, e.g. T. reesei, T. viride;Yersinia, e.g. Y. enterocolitica. In one embodiment, the lactase is anintracellular component of microorganisms like Kluyveromyces andBacillus. Kluyveromyces, especially K. fragilis and K. lactis, and otherfungi such as those of the genera Candida, Torula and Torulopsis, are acommon source of fungal lactases, whereas B. coagulans and B circulansare well known sources for bacterial lactases. Several commerciallactase preparations derived from these organisms are available such asLactozym® (available from Novozymes, Denmark), HA-Lactase (availablefrom Chr. Hansen, Denmark) and Maxilact® (available from DSM, theNetherlands), all from K. lactis. All these lactases are so calledneutral lactases having a pH optimum between pH 6 and pH 8. When suchlactases are used in the production of, e.g., low-lactose yoghurt, theenzyme treatment will either have to be done in a separate step beforefermentation or rather high enzyme dosages have to be used, becausetheir activity drop as the pH decreases during fermentation. Also, theselactases are not suitable for hydrolysis of lactose in milk performed athigh temperature, which would in some cases be beneficial in order tokeep the microbial count low and thus ensure good milk quality.

In one embodiment, the enzyme is a lactase from a bacterium, e.g. fromthe family Bifidobacteriaceae, such as from the genus Bifidobacteriumsuch as the lactase described in WO 2009/071539.

Further Aspects of the Invention

-   Aspect 1. A polypeptide having transgalactosylating activity, which    comprises an amino acid sequence having at least 90% sequence    identity with SEQ ID NO: 1, and wherein said polypeptide, when being    an expression product in a Bacillus subtilis strain BG3594 of a    nucleic acid sequence, which encodes said polypeptide, is the only    polypeptide expression product of said nucleic acid sequence that    exhibits transgalactosylating activity.-   Aspect 2. A polypeptide having transgalactosylating activity    selected from the group consisting of:    -   a. a polypeptide comprising an amino acid sequence having at        least 90% sequence identity with SEQ ID NO: 1, wherein said        polypeptide consists of at most 980 amino acid residues,    -   b. a polypeptide comprising an amino acid sequence having at        least 97% sequence identity with SEQ ID NO: 2, wherein said        polypeptide consists of at most 975 amino acid residues,    -   c. a polypeptide comprising an amino acid sequence having at        least 96.5% sequence identity with SEQ ID NO: 3, wherein said        polypeptide consists of at most 1300 amino acid residues,    -   d. a polypeptide encoded by a polynucleotide that hybridizes        under at least low stringency conditions with i) the nucleic        acid sequence comprised in SEQ ID NO: 9, 10, 11, 12 or 13        encoding the polypeptide of SEQ ID NO: 1, 2, 3, 4, or 5; or ii)        the complementary strand of i),    -   e. a polypeptide encoded by a polynucleotide comprising a        nucleotide sequence having at least 70% identity to the        nucleotide sequence encoding for the polypeptide of SEQ ID NO:        1, 2, 3, 4 or 5 or the nucleotide sequence comprised in SEQ ID        NO: 9, 10, 11, 12 or 13 encoding a mature polypeptide, and    -   f. a polypeptide comprising a deletion, insertion and/or        conservative substitution of one or more amino acid residues of        SEQ ID NO: 1, 2, 3, 4 or 5.-   Aspect 3. A polypeptide having transgalactosylating activity    selected from the group consisting of:    -   a. a polypeptide comprising an amino acid sequence having at        least 90% sequence identity with SEQ ID NO: 1, wherein said        polypeptide consists of at most 980 amino acid residues,    -   b. a polypeptide comprising an amino acid sequence having at        least 96.5% sequence identity with SEQ ID NO: 3, wherein said        polypeptide consists of at most 1300 amino acid residues,    -   c. a polypeptide encoded by a polynucleotide that hybridizes        under at least low stringency conditions with i) the nucleic        acid sequence comprised in SEQ ID NO: 9, 10, 11, 12 or 13        encoding the polypeptide of SEQ ID NO: 1, 2, 3, 4, or 5; or ii)        the complementary strand of i),    -   d. a polypeptide encoded by a polynucleotide comprising a        nucleotide sequence having at least 70% identity to the        nucleotide sequence encoding for the polypeptide of SEQ ID NO:        1, 2, 3, 4 or 5 or the nucleotide sequence comprised in SEQ ID        NO: 9, 10, 11, 12 or 13 encoding a mature polypeptide, and    -   e. a polypeptide comprising a deletion, insertion and/or        conservative substitution of one or more amino acid residues of        SEQ ID NO: 1, 2, 3, 4 or 5.-   Aspect 4. The polypeptide according to any one of the preceding    aspects, wherein said polypeptide, when being an expression product    in a Bacillus subtilis strain BG3594 of a nucleic acid sequence,    which encodes said polypeptide, is the only polypeptide expression    product of said nucleic acid sequence that exhibits    transgalactosylating activity.-   Aspect 5. The polypeptide according to any one of the preceding    aspects comprising an amino acid sequence having at least 96.5%    sequence identity with SEQ ID NO: 3, wherein said polypeptide    consists of at most 1300 amino acid residues.-   Aspect 6. The polypeptide according to any one of the preceding    aspects comprising an amino acid sequence having at least 90%    sequence identity with SEQ ID NO: 1, wherein said polypeptide    consists of at most 980 amino acid residues.-   Aspect 7. The polypeptide according to any of aspects 1, 2 and 4    comprising an amino acid sequence having at least 97% sequence    identity with SEQ ID NO: 2, wherein said polypeptide consists of at    most 975 amino acid residues.-   Aspect 8. A polypeptide comprising an amino acid sequence having at    least 90% sequence identity with SEQ ID NO: 1, wherein said    polypeptide consists of at most 980 amino acid residues.-   Aspect 9. The polypeptide according to aspect 8, wherein said    polypeptide has transgalactosylating activity.-   Aspect 10. The polypeptide according to any one of aspects 8-9,    wherein said polypeptide, when being an expression product in a    Bacillus subtilis strain BG3594 of a nucleic acid sequence, which    encodes said polypeptide, is the only polypeptide expression product    of said nucleic acid sequence that exhibits transgalactosylating    activity.-   Aspect 11. The polypeptide according to any one of the preceding    aspects, wherein said sequence identity is at least 95%, such as,    e.g. at least 96%, at least 97%, at least 98%, at least 99% or at    least 100% sequence identity.-   Aspect 12. A polypeptide wherein said polypeptide has at least 90%    sequence identity with SEQ ID NO: 1.-   Aspect 13. The polypeptide according to aspect 12, wherein said    polypeptide has transgalactosylating activity.-   Aspect 14. The polypeptide according to any one of the preceding    aspects, wherein said polypeptide has at least 90% such as, e.g. at    least 91%, at least 92%, at least 93%, at least 94%, at least 95%,    at least 96%, at least 97%, at least 98%, or at least 99% sequence    identity with SEQ ID NO: 1.-   Aspect 15. The polypeptide according to any one of aspects 1-14,    wherein the degree of sequence identity between a query sequence and    a reference sequence is determined by 1) aligning the two sequences    by any suitable alignment program using the default scoring matrix    and default gap penalty, 2) identifying the number of exact matches,    where an exact match is where the alignment program has identified    an identical amino acid or nucleotide in the two aligned sequences    on a given position in the alignment and 3) dividing the number of    exact matches with the length of the reference sequence.-   Aspect 16. The polypeptide according to any one of aspects 1-15,    wherein the degree of sequence identity between a query sequence and    a reference sequence is determined by 1) aligning the two sequences    by any suitable alignment program using the default scoring matrix    and default gap penalty, 2) identifying the number of exact matches,    where an exact match is where the alignment program has identified    an identical amino acid or nucleotide in the two aligned sequences    on a given position in the alignment and 3) dividing the number of    exact matches with the longest of the two sequences.-   Aspect 17. The polypeptide according to any one of aspects 1-16,    wherein the degree of sequence identity between a query sequence and    a reference sequence is determined by 1) aligning the two sequences    by any suitable alignment program using the default scoring matrix    and default gap penalty, 2) identifying the number of exact matches,    where an exact match is where the alignment program has identified    an identical amino acid or nucleotide in the two aligned sequences    on a given position in the alignment and 3) dividing the number of    exact matches with the “alignment length”, where the alignment    length is the length of the entire alignment including gaps and    overhanging parts of the sequences.-   Aspect 18. The polypeptide according to any one of aspects 15-17,    wherein the suitable alignment program is a global alignment    program.-   Aspect 19. The polypeptide according to aspect 18, wherein the    global alignment program uses the Needleman-Wunsch algorithm.-   Aspect 20. The polypeptide according to any of aspects 18 and 19,    wherein the global alignment program is selected from the group    consisting of EMBOSS Needle and EMBOSS stretcher.-   Aspect 21. The polypeptide according to any one of the preceding    aspects, which consists of at most 975 amino acid residues, such as,    e.g. at most 970 amino acid residues, such as at most 950 amino acid    residues, such as at most 940 amino acid residues, at most 930 amino    acid residues, at most 920 amino acid residues, at most 910 amino    acid residues, at most 900 amino acid residues, at most 895 amino    acid residues or at most 890 amino acid residues.-   Aspect 22. The polypeptide according to any one of the preceding    aspects, which consists of 887 amino acid residues.-   Aspect 23. The polypeptide according to any one of the preceding    aspects, which comprises SEQ ID NO:1.-   Aspect 24. The polypeptide according to any one of the preceding    aspects, which consist of the amino acid sequence of SEQ ID NO:1.-   Aspect 25. The polypeptide according to any one of the preceding    aspects, wherein said polypeptide consists of 965 amino acid    residues.-   Aspect 26. The polypeptide according to any of aspects 1-23 and 25,    wherein said polypeptide has at least 96.5% sequence identity to SEQ    ID NO: 2.-   Aspect 27. The polypeptide according to any of aspects 1-23 and    25-26, wherein said polypeptide has at least 97%, such as, e.g. at    least 98% or at least 99% sequence identity with SEQ ID NO: 2.-   Aspect 28. The polypeptide according to any one of aspects 1-23 and    25-27, wherein said polypeptide comprises SEQ ID NO: 2.-   Aspect 29. The polypeptide according to any one of aspects 1-23 and    25-28, wherein said polypeptide consists of the amino acid sequence    of SEQ ID NO: 2.-   Aspect 30. A polypeptide comprising an amino acid sequence having at    least 96.5% sequence identity with SEQ ID NO: 3, wherein said    polypeptide consists of at most 1300 amino acid residues.-   Aspect 31. The polypeptide according to aspect 30, wherein said    polypeptide has transgalactosylating activity.-   Aspect 32. The polypeptide according to any one of aspects 30-31,    wherein said polypeptide, when being an expression product in a    Bacillus subtilis strain BG3594 of a nucleic acid sequence, which    encodes said polypeptide, is the only polypeptide expression product    of said nucleic acid sequence that exhibits transgalactosylating    activity.-   Aspect 33. The polypeptide according to any one of aspects 1-5,    15-20 and 30-32, wherein said sequence identity is at least 97%,    such as, e.g. at least 98%, at least 99% or at least 100% sequence    identity.-   Aspect 34. The polypeptide according to any one of aspects 1-5,    15-20 and 30-33, which comprises SEQ ID NO: 3.-   Aspect 35. The polypeptide according to any one of aspects 11-5,    15-20 and 30-34, which consists of the amino acid sequence of SEQ ID    NO: 3.-   Aspect 36. The polypeptide according to any one of aspects 1-5,    15-20 and 30-34, which consists of at most 1290 amino acid residues,    such as, e.g. at most 1280, at most 1270, at most 1260, at most    1250, at most 1240, at most 1230, at most 1220 or at most 1215 amino    acid residues.-   Aspect 37. The polypeptide according to any one of aspects 1-5,    15-20, 30-34 and 36, which consists of 1211 amino acid residues.-   Aspect 38. The polypeptide according to any one of aspects 1-5,    15-20, 30-34 and 36-37, wherein said polypeptide has at least 98.5%    sequence identity with SEQ ID NO: 5.-   Aspect 39. The polypeptide according to any one of aspects 1-5,    15-20, 30-34 and 36-38, wherein said polypeptide has at least 99% or    at least 99.5% sequence identity with SEQ ID NO: 5.-   Aspect 40. The polypeptide according to any one of aspects 1-5,    15-20, 30-34 and 36-39, which comprises SEQ ID NO: 5.-   Aspect 41. The polypeptide according to any one of aspects 1-5,    15-20, 30-34 and 36-40, which consists of the amino acid sequence of    SEQ ID NO: 5.-   Aspect 42. The polypeptide according to any one of aspects 1-5,    15-20, 30-34 and 36-40, which consists of at most 1210 amino acid    residues, such as, e.g. at most 1200, at most 1190, at most 1180, at    most 1170, at most 1160, at most 1150 or at most 1145 amino acid    residues.-   Aspect 43. The polypeptide according to any one of aspects 1-5,    15-20, 30-34, 36-40 and 42, which consists of 1142 amino acid    residues.-   Aspect 44. The polypeptide according to any one of aspects 1-5,    15-20, 30-34, 36-40 and 42-43, wherein said polypeptide has at least    96% sequence identity with SEQ ID NO: 4.-   Aspect 45. The polypeptide according to any one of aspects 1-5,    15-20, 30-34, 36-40 and 42-44, wherein said polypeptide has at least    97%, such as, e.g., at least 98% or at least 99% sequence identity    with SEQ ID NO: 4.-   Aspect 46. The polypeptide according to any one of aspects 1-5,    15-20, 30-34, 36-40 and 42-45, which comprises SEQ ID NO: 4.-   Aspect 47. The polypeptide according to any one of aspects 1-5,    15-20, 30-34, 36-40 and 42-46, which consists of the amino acid    sequence of SEQ ID NO: 4.-   Aspect 48. The polypeptide according to any one aspects 1-5, 15-20,    30-34, 36-40 and 42-46, which consists of at most 1130 amino acid    residues, such as, e.g. at the most 1120, at the most 1110, at the    most 1100, at the most 1090, at the most 1080, at the most 1070, at    the most 1060, at the most 1050, at the most 1055 or at the most    1040 amino acid residues.-   Aspect 49. The polypeptide according to any one of aspects 1-5,    15-20, 30-34, 36-40, 42-46 and 48, wherein said polypeptide consists    of 1038 amino acid residues.-   Aspect 50. The polypeptide according to any one of aspects 1-5,    15-20, 30-34, 36-40, 42-46 and 48-49, wherein said polypeptide has    at least 96.5% sequence identity with SEQ ID NO: 3.-   Aspect 51. The polypeptide according to any one of aspects 1-5,    15-20, 30-34, 36-40, 42-46 and 48-50, wherein said polypeptide has    at least 97%, such as, e.g., at least 98% or at least 99% sequence    identity with SEQ ID NO: 3.-   Aspect 52. The polypeptide according to any one of aspects 1-5,    15-20, 30-34, 36-40, 42-46 and 48-51, which comprises SEQ ID NO:3.-   Aspect 53. The polypeptide according to any one of aspects 1-5,    15-20, 30-34, 36-40, 42-46 and 48-52, which consists of the amino    acid sequence of SEQ ID NO: 3.-   Aspect 54. The polypeptide according to any of the preceding    aspects, wherein the ratio of transgalactosylating    activity:β-galactosidase activity is at least 0.5, such as at least    1, such as at least 1.5, such as at least 2 after 30 min. reaction    such as above a concentration of 3% w/w initial lactose    concentration.-   Aspect 55. The polypeptide according to any one of the preceding    aspects, wherein the ratio of transgalactosylating    activity:β-galactosidase activity is at least 2.5, such as at least    3, such as at least 4, such as at least 5, such as at least 6, such    as at least 7, such as at least 8, such as at least 9, such as at    least 10, such as at least 11, or such as at least 12 after 30 min.    reaction such as above a concentration of 3% w/w initial lactose    concentration.-   Aspect 56. The polypeptide according to any one of aspects 54-55,    wherein the initial lactose concentration is 3% w/w.-   Aspect 57. The polypeptide according to any one of the preceding    aspects, which is isolated and/or purified.-   Aspect 58. The polypeptide according to any one of the preceding    aspects, which is recombinantly produced.-   Aspect 59. The polypeptide according to any one of the preceding    aspects, wherein said polypeptide has a ratio of    transgalactosylation activity above 100%.-   Aspect 60. The polypeptide according to any one of the preceding    aspects, wherein said polypeptide has a ratio of    transgalactosylation activity above 150%, 175% or 200%.-   Aspect 61. The polypeptide according to any one of the preceding    aspects, having a glycoside hydrolase catalytic core with an amino    acid sequence selected from the group consisting of SEQ ID NO:7.-   Aspect 62. The polypeptide according to any one of the preceding    aspects containing a Glyco_hydro2N (PF02837), a Glyco_hydro    (PF00703) and/or a Glyco_hydro 2C (PF02836) domains.-   Aspect 63. The polypeptide according to any one of the preceding    aspects containing the Bacterial Ig-like domain (group 4) (PF07532).-   Aspect 64. The polypeptide according to any one of the preceding    aspects, which is derived from Bifidobacterium bifidum.-   Aspect 65. The polypeptide according to any one of the preceding    aspects having a pH optimum of 6.5-7.5.-   Aspect 66. The polypeptide according to any one of the preceding    aspects having a temperature optimum of 30-60 such as 42-60 degree    celcius.-   Aspect 67. The polypeptide according to any one of the preceding    aspects, wherein the percentage of identity of one amino acid    sequence with, or to, another amino acid sequence is determined by    the use of Blast with a word size of 3 and with BLOSUM 62 as the    substitution matrix.-   Aspect 68. The polypeptide according to any one of the preceding    aspects, which polypeptide is a recombinant polypeptide.-   Aspect 69. The polypeptide according to any one of the preceding    aspects, which polypeptide is freeze-dried.-   Aspect 70. The polypeptide according to any one of the preceding    aspects, which polypeptide is in solution.-   Aspect 71. The polypeptide according to any one of the preceding    aspects, which polypeptide is isolated.-   Aspect 72. The polypeptide according to any one of the preceding    aspects, which polypeptide is purified.-   Aspect 73. A nucleic acid capable of encoding a polypeptide    according to any one of the preceding aspects.-   Aspect 74. An expression vector comprising a nucleic acid according    to aspect 73, or capable of expressing a polypeptide according to    any one aspects 1-72.-   Aspect 75. A cell capable of expressing a polypeptide according to    any one of aspects 1-72.-   Aspect 76. A method of expressing a polypeptide, the method    comprising obtaining a cell according aspect 75 and expressing the    polypeptide from the cell, and optionally purifying the polypeptide.-   Aspect 77. A composition comprising a polypeptide as defined in any    one of aspects 1-72, preferably a food composition, more preferably    a dairy product.-   Aspect 78. The composition according to aspect 77 comprising at    least 5%, such as e.g. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%    w/w of one or more polypeptide(s) as defined in any one of aspects    1-72 based on the total amount of polypeptides in the composition    having at least 70%, e.g. such as 72%, 74%, 74%, 78%, 80%, 82%, 84%,    86%, 88%, 90% sequence identity with SEQ ID NO: 22.-   Aspect 79. The composition according to any one of aspects 77-78,    wherein the one or more polypeptide(s) is selected from the group    consisting of a polypeptide consisting of SEQ ID NO: 1, 2, 3, 4 and    5.-   Aspect 80. The composition according to any one of aspects 77-79,    wherein the one or more polypeptide(s) is selected from the group    consisting of a polypeptide consisting of SEQ ID NO: 1, 2, and 3.-   Aspect 81. The composition according to any one of aspects 77-80,    wherein the one or more polypeptide(s) is a polypeptide consisting    of SEQ ID NO: 1 or 2.-   Aspect 82. A method for producing a food product by treating a    substrate comprising lactose with a polypeptide as defined in any on    of aspects 1-72.-   Aspect 83. The method according to aspect 82 for producing a dairy    product by treating a milk-based substrate comprising lactose with a    polypeptide as defined in any one of aspects 1-72.-   Aspect 84. The method according to any one of aspects 82-83 further    treating the substrate with a hydrolysing beta-galactosidase.-   Aspect 85. A galacto-oligosaccharide or composition thereof obtained    by treating a substrate comprising lactose with a polypeptide as    defined in any one of aspects 1-72.-   Aspect 86. A nucleic acid capable of encoding a polypeptide    according to any one of the aspects 1-72.-   Aspect 87. The nucleic acid according to aspect 86 having a nucleic    acid sequence which is at least 60% identical to SEQ ID NO: 9, 10,    11, 12 or 13.-   Aspect 88. A plasmid comprising a nucleic acid according to any one    of the aspects 86-87.-   Aspect 89. An expression vector comprising a nucleic acid according    to any one of the aspects 86-87, or capable of expressing a    polypeptide according to any one of the aspects 1-72.-   Aspect 90. A host cell comprising, preferably transformed with, a    plasmid according to aspect 88 or an expression vector according to    aspect 89.-   Aspect 91. A cell capable of expressing a polypeptide according to    any one of the aspects 1-72.-   Aspect 92. The host cell according to aspect 90, or the cell    according to aspect 91, which is a bacterial, fungal or yeast cell.-   Aspect 93. The cell according to aspect 92, wherein the cell is    selected from the group consisting of Ruminococcus, Bifidobacterium,    Lactococcus, Lactobacillus, Streptococcus, Leuconostoc, Escherichia,    Bacillus, Streptomyces, Saccharomyces, Kluyveromyces, Candida,    Torula, Torulopsis and Aspergillus.-   Aspect 94. The cell according to aspect 93, wherein the cell is    selected from the group consisting of Ruminococcus hansenii,    Ruminococcus lactaris, Bifidobacterium breve, Bifidobacterium    longum, Bifidobacterium infantis, Bifidobacterium bifidum and    Lactococcus lactis.-   Aspect 95. A method of expressing a polypeptide, the method    comprising obtaining a host cell or a cell according to any one of    aspects 91-94 and expressing the polypeptide from the cell or host    cell, and optionally purifying the polypeptide.-   Aspect 96. A composition comprising a polypeptide as defined in any    of aspects 1-72 and a stabilizer.-   Aspect 97. A composition comprising a polypeptide as defined in any    of aspects 1-72 and a carbohydrate substrate.-   Aspect 98. The composition according to aspect 97, wherein the    carbohydrate substrate is a disaccharide.-   Aspect 99. The composition according to aspect 98, wherein the    disaccharide is lactose.-   Aspect 100. A method for producing a dairy product by treating a    milk-based substrate comprising lactose with a polypeptide according    to any one of aspects 1-72.-   Aspect 101. The method according to aspect 90, wherein the    polypeptide has a ratio of transgalactosylation activity as defined    above.-   Aspect 102. The method according to any one of aspects 100-101,    wherein the milk-based substrate is yoghurt, cheese, or fermented    dairy products.-   Aspect 103. The method according to any one of aspects 100-102,    further comprising fermenting said substrate with a microorganism    capable of fermenting said substrate.-   Aspect 104. The method according to aspect 103, wherein the    milk-based substrate is yogurt.-   Aspect 105. The method according to any one of aspects 100-104,    wherein the treatment with the polypeptide and the microorganism is    performed essentially at the same time.-   Aspect 106. The method according to any one of aspects 100-105,    wherein the polypeptide and the microorganism are added to the    milk-based substrate essentially at the same time.-   Aspect 107. Use of a cell of any of above aspects for producing a    product selected from the group consisting of yoghurt, cheese,    fermented milk product, dietary supplement and probiotic comestible    product.-   Aspect 108. A dairy product comprising a cell of any of above    aspects.-   Aspect 109. A dairy product comprising a polypeptide as defined in    any one of aspects 1-72.-   Aspect 110. A dairy product comprising a polypeptide as defined in    any one of aspects 1-72 in a concentration of 0.01-1000 ppm.-   Aspect 111. A dairy product comprising an inactivated polypeptide as    defined in any one of aspects 1-72.-   Aspect 112. A dairy product comprising an inactivated polypeptide as    defined in any one of aspects 1-72 in a concentration of 0.01-1000    ppm.-   Aspect 113. A dairy product comprising GOS formed in situ by a    polypeptide as defined in any one of aspects 1-72.-   Aspect 114. Use of a transgalactosylating polypeptide of any one of    aspects 1-72 or a cell of any one of above aspects, for producing    galacto-oligosaccharides.-   Aspect 115. Use of a transgalactosylating polypeptide of any one of    aspects 1-72 or a cell of any one of above aspects, for producing    galacto-oligosaccharides to be part of a product selected from the    group consisting of yoghurt, cheese, fermented dairy products,    dietary supplements and probiotic comestible products.-   Aspect 116. Use of a transgalactosylating polypeptide of any one of    aspects 1-72 or a cell of any one of above aspects, for producing    galacto-oligosaccharides to enhance the growth of Bifidobacterium.-   Aspect 117. Use of a transgalactosylating polypeptide of any one of    aspects 1-72 or a cell of any one of above aspects, for producing    galacto-oligosaccharides to enhance the growth of Bifidobacterium in    a mixed culture fermentation.-   Aspect 118. A process for producing a transgalactosylating    polypeptide of any one of aspects 1-72, comprising culturing a cell    of any one of above aspects in a suitable culture medium under    conditions permitting expression of said polypeptide, and recovering    the resulting polypeptide from the culture.-   Aspect 119. A process for producing galacto-oligosaccharides,    comprising contacting of an polypeptide of any one of aspects 1-72    or a cell of any one of above aspects with a milk-based solution    comprising lactose.-   Aspect 120. A polypeptide which is a C-terminal truncated fragment    of SEQ ID NO:22 having transgalactosylating activity and which are    stable against further truncation such as by proteolytic degradation    when produced in a suitable organism such as Bacillus subtilis e.g.    Bacillus subtilis strain BG3594 and/or which are stable against    further truncation during storage after final formulation.-   Aspect 121. The polypeptide according to aspect 120 which is as    further defined in any one of aspects 1-72.

Materials and Methods Method 1 Production of Polypeptide

Synthetic genes designed to encode the Bifidobacterium bifidum fulllength (1752 residues) gene with codons optimised for expression inBacillus subtilis were purchased from GeneART (Regensburg, Germany) SEQID No. 8

The Bifidobacterium bifidum truncation mutants were constructed usingpolymerase chain reaction with reverse primers that allowed specificamplification of the selected region of the synthetic gene.

Forward primer: GGGGTAACTAGTGGAAGATGCAACAAGAAG (SpeI underlined). (SEQID NO: 15) Reverse primers: Truncation mutant Primer sequence BIF917(SEQ ID NO: 9) GCGCTTAATTAATTATGTTTTTTCTGTGCTTGTTC SEQ ID NO: 16 BIF995(SEQ ID NO: 10) GCGCTTAATTAATTACAGTGCGCCAATTTCATCAATCA SEQ ID NO: 17BIF1068 (SEQ ID NO: 11) GCGCTTAATTAATTATTGAACTCTAATTGTCGCTG SEQ ID NO:18 BIF1241 (SEQ ID NO: 12) GCGCTTAATTAATTATGTCGCTGTTTTCAGTTCAAT SEQ IDNO: 19 BIF1326 (SEQ ID NO: 13) GCGCTTAATTAATTAAAATTCTTGTTCTGTGCCCA SEQID NO: 20 BIF1478 (SEQ ID NO: 14) GCGCTTAATTAATTATCTCAGTCTAATTTCGCTTGCGCSEQ ID NO: 21

The synthetic gene was cloned into the pBNspe Bacillus subtilisexpression vector using the unique restriction sites SpeI and Pad(FIG. 1) and the isolated plasmids were transformed into the Bacillussubtilis strain BG3594. Transformants were restreaked onto LB platescontaining 10 μg/mL Neomycin as selection.

A preculture was setup in LB media containing 10 μg/mL Neomycin andcultivated for 7 hours at 37° C. and 180 rpm shaking. 500 μL of thispreculture was used to inoculate 50 mL Grant's modified mediumcontaining 10 μg/mL Neomycin at allowed to grow for 68 hours at 33° C.and 180 rpm shaking.

Cells were lysed by addition directly to the culture media of 1 mg/mlLysozyme (Sigma-Aldrich) and 10 U/ml Benzonase (Merck) finalconcentrations and incubated for 1 hr at 33° C. at 180 RPM. Lysates werecleared by centrifugation at 10.000×g for 20 minutes and subsequentlysterile filtered.

Grant's Modified Media was Prepared According to the FollowingDirections:

PART I (Autoclave) Soytone 10 g Bring to 500 mL per liter PART II 1MK₂HPO₄ 3 mL Glucose 75 g Urea 3.6 g Grant's 10X MOPS 100 mL Bring to 400mL per liter

PART I (2 w/w % Soytone) was prepared, and autoclaved for 25 minutes at121° C.

PART II was prepared, and mixed with PART 1 and pH was adjusted to pH to7.3 with HCl/NaOH.

The volume was brought to full volume and sterilized through 0.22-μm PESfilter.

10×MOPS Buffer was Prepared According to the Following Directions:

83.72 g Tricine 7.17 g KOH Pellets 12 g NaCl 29.22 g 0.276M K2SO4 10 mL0.528M MgCl2 10 mL Grant's Micronutrients 100X

Bring to app. 900 mL with water and dissolve. Adjust pH to 7.4 with KOH,fill up to 1 L and sterile filter the solution through 0.2 μm PESfilter.

100× Micronutrients was Prepared According to the Following Directions:

Sodium Citrate•2H2O 1.47 g CaCl2•2H2O 1.47 g FeSO4•7H2O 0.4 g MnSO4•H2O0.1 g ZnSO4•H2O 0.1 g CuCl2•2H2O 0.05 g CoCl2•6H2O 0.1 g Na2MoO4•2H2O0.1 g

Dissolve and adjust volume to 1 L with water.

Sterilization was through 0.2 μm PES filter.

Storing was at 4° C. avoid light.

Method 2 Purification and Enzyme Preparations

The filtrated enzyme isolate was concentrated using a VivaSpin ultrafiltration device with a 10 kDa MW cut off (Vivaspin 20, Sartorius,Lot#12VS2004) and the concentrate was loaded onto a PD10 desaltingcolumn (GE healthcare, Lot#6284601) and eluted in 20 mM Tris-HCl pH 8.6.Chromatography was carried out manually on an Äkta FPLC system (GEHealthcare). 4 mL of the desalted sample, containing approximately 20 mgprotein, was loaded onto a 2 mL HyperQ column (HyperCel™, Q sorbent)equilibrated with 20 mM Tris-HCl pH 8.6 at a flowrate of 1 ml/min. Thecolumn was thoroughly washed with 30 CV (column volumes) wash buffer andthe bound β-galactosidase was eluted with a 100CV long gradient into 20mM Tris-HCl pH 8.6 250 mM NaCl. Remaining impurities on the column wereremoved with a one-step elution using 20 mM Tris-HCl pH 8.6 500 mM NaCl.Protein in the flow through and elution was analyzed for β-galactosidaseactivity and by SDS-page.

SDS-page gels were run with the Invitrogen NuPage® Novex 4-12% Bis-Trisgel 1.0 mm, 10 well (Cat#NP0321box), See-Blue® Plus2 prestained Standard(Cat# LC5925) and NuPAGE® MES SDS Running Buffer (Cat# NP0002) accordingto the manufacturer's protocol. Gels were stained with Simply BlueSafestain (Invitrogen, Cat# LC6060) (FIG. 2).

Method 3 Measuring β-Galactosidase Activity

Enzymatic activity was measured using the commercially availablesubstrate 2-Nitrophenyl-β-D-Galactopyranoside (ONPG) (Sigma N1127).

ONPG w/o acceptor 100 mM KPO4 pH 6.0 12.3 mM ONPG ONPG supplemented withacceptor 100 mM KPO4 pH 6.0 20 mM Cellobiose 12.3 mM ONPG STOP Solution10% Na₂CO₃

10 μl dilution series of purified enzyme was added in wells of amicrotiter plates containing 90 μl ONPG-buffer with or without acceptor.Samples were mixed and incubated for 10 min at 37° C., subsequently 100μl STOP Solution were added to each well to terminate reaction.Absorbance measurements were recorded at 420 nm on a Molecular DeviceSpectraMax platereader controlled by the Softmax software package.

The ratio of transgalactosylation activity was calculated as follows

Ratio of transgalctosylationactivity=(Abs420^(+Cellobiose)/Abs420^(−Cellobiose))*100,

for dilutions where the absorbance was between 0.5 and 1.0 (FIG. 3).

Method 4 Determination of LAU Activity Principle:

The principle of this assay method is that lactase hydrolyzes2-o-nitrophenyl-β-D-galactopyranoside (ONPG) into 2-o-nitrophenol (ONP)and galactose at 37° C. The reaction is stopped with the sodiumcarbonate and the liberated ONP is measured in spectrophotometer orcolorimeter at 420 nm.

Reagents:

MES buffer pH 6.4 (100 mM MES pH 6.4, 10 mM CaCl₂): Dissolve 19.52 g MEShydrate (Mw: 195.2 g/mol, Sigma-aldrich #M8250-250G) and 1.470 g CaCl₂di-hydrate (Mw: 147.01 g/mol, Sigma-aldrich) in 1000 ml ddH₂O, adjust pHto 6.4 by 10M NaOH. Filter the solution through 0.2 μm filter and storeat 4° C. up to 1 month.

ONPG substrate pH 6.4 (12.28 mM ONPG, 100 mM MES pH 6.4, 10 mM CaCl₂):Dissolve 0.370 g 2-o-nitrophenyl-β-D-galactopyranoside (ONPG, Mw: 301.55g/mol, Sigma-aldrich #N1127) in 100 ml MES buffer pH 6.4 and store darkat 4° C. for up to 7 days.

Stop reagent (10% Na₂CO₃): Dissolve 20.0 g Na₂CO₃ in 200 ml ddH₂O,Filter the solution through 0.2 μm filter and store at RT up to 1 month.

Procedure:

Dilution series of the enzyme sample was made in the MES buffer pH 6.4and 10 μL of each sample dilution were transferred to the wells of amicrotiter plate (96 well format) containing 90 μl ONPG substrate pH6.4. The samples were mixed and incubated for 5 min at 37° C. using aThermomixer (Comfort Thermomixer, Eppendorf) and subsequently 100 μlStop reagent was added to each well to terminate the reaction. A blankwas constructed using MES buffer pH 6.4 instead of the enzyme sample.The increase in absorbance at 420 nm was measured at a ELISA reader(SpectraMax platereader, Molecular Device) against the blank.

Calculation of Enzyme Activity:

The molar extinction coefficient of 2-o-nitrophenol (Sigma-aldrich#33444-25G) in MES buffer pH 6.4 was determined (0.5998×10⁻⁶M⁻¹×cm⁻¹).One unit (U) of lactase activity (LAU) was defined as that correspondingto the hydrolysis of 1 nmol of ONPG per minute. Using microtitre plateswith a total reaction volume of 200 μL (light path of 0.52 cm) thelactase activity per mL of the enzyme sample may be calculated using thefollowing equation:

${{LAU}\text{/}{{ml}( \frac{nmol}{\min \cdot {mL}} )}} = \frac{{Abs}_{420} \times 200\mspace{11mu} {µL} \times {dilution}\mspace{14mu} {factor}}{{0.5998 \cdot 10^{3} \cdot {nM}^{- 1} \cdot {cm}^{- 1}} \times 0.52{\; \mspace{11mu}}{cm} \times 5\mspace{14mu} \min \times 0.01\mspace{14mu} {mL}}$

Calculation of Specific Activity for BIF917 Shown Herein as SEQ ID NO:1: Determination of BIF917 Concentration:

Quantification of the target enzyme (BIF917) and truncation productswere determined using the Criterion Stain free SDS-page system (BioRad).Any kD Stain free precast Gel 4-20% Tris-HCl, 18 well (Comb #345-0418)was used with a Serva Tris-Glycine/SDS buffer (BioRad cat. #42529). Gelswere run with the following parameters: 200 V, 120 mA, 25 W, 50 min. BSA(1.43 mg/ml) (Sigma-Aldrich, cat. #500-0007) was used as proteinstandard and Criterion Stain Free Imager (BioRad) was used with ImageLab software (BioRad) for quantification using band intensity withcorrelation of the tryptophan content.

The specific LAU activity of BIF917 was determined from crude ferment(ultra filtration concentrate) of two independent fermentations (asdescribed in method 1) and using 5 different dilutions (see table 1).

The specific activity of BIF917 was found to be 21.3 LAU/mg or 0.0213LAU/ppm.

TABLE 1 Determination of BIF917 specific activity Protein Protein(BIF917) (BIF917) Specific Specific Sample Dilution Activityconcentration concentration activity activity ID Enzyme Fermentationfactor LAU/ml mg/ml ppm LAU/mg LAU/ppm 1 BIF 917 a 5 26.9 1.23 1232 21.90.0219 2 BIF 917 a 10 53.9 2.44 2437 22.1 0.0221 3 BIF 917 a 10 75.43.56 3556 21.2 0.0212 4 BIF 917 a 20 163.9 7.78 7778 21.1 0.0211 5 BIF917 a 30 233.6 11.06 11065 21.1 0.0211 6 BIF 917 b 5 30.26825 1.34 134222.6 0.0226 7 BIF 917 b 10 55.91536 2.61 2607 21.4 0.0214 8 BIF 917 b 1076.96056 3.70 3697 20.8 0.0208 9 BIF 917 b 20 156.986 7.75 7755 20.20.0202 10 BIF 917 b 30 236.9734 11.45 11452 20.7 0.0207 Arg 21.3 0.0213Std 0.700976 0.000701

EXAMPLES Example 1 Determining β-Galactosidase Activity of BIFTruncation Variants

Eight different truncation variants: BIF_(—)917, BIF_(—)995,BIF_(—)1068, BIF_(—)1172, BIF_(—)1241, BIF1326, BIF_(—)1400 andBIF_(—)1478 were constructed as described using method 1 and purified asdescribed in method 2 (see FIG. 2).

The β-galactosidase activity was determined of all truncation variantsin presence and absence of cellobiose using the described method 3above.

Results

The ratio of transgalactosylation activity((Abs420^(+Cellobiose)/Abs420^(−Cellobiose))*100) was calculated fromthe measured β-galactosidase activity for each variant and is shown inFIG. 3. Variants having a length of 1241 residues or less shows a ratioof transgalactosylation activity above 100%, indicating that thesevariants are predominantly transgalactosylating. The variants with alength that is more that 1241 residues shows a ratio oftransgalactosylation activity below 100%, indicating that these variantsare predominantly hydrolytic. BIF_(—)917 and BIF_(—)995 have the highestratio of transgalactosylation activity around 250%.

Example 2 GOS Generated in a Yoghurt Matrix

Evaluation of BIF enzymes in GOS production were tested in an yogurtapplication mimic. Batch experiments with a volume of 100 μl wereperformed in 96 well MTP plates using a yogurt mix, consisting of98.60%(w/v) fresh pasteurized low-fat milk (Mini-mælk, Arla Foods,Denmark) and 1.4% (w/v) Nutrilac YQ-5075 whey ingredient (Arla). Tocompletely hydrate Nutrilac YQ-5075 the mixture was left with agitationfor 20 h and afterwards added 20 mM NaPhosphate pH 6.5 to ensure a pH of6.5. This milk-base was used plain and the lactose concentration wasdetermined to be 5.5%(w/v), corresponding to 5.3% (w/w) in thissolution. The following correlation is valid in the present example:1%(w/v) lactose=0.9587%(w/w) lactose. 90 μl of the milk-base was mixedwith 10 μl of the purified enzymes, sealed with tape and incubated at43° C. for 3 hours. The reaction was stopped by 100 μl 10% Na₂CO₃.Samples stored at −20° C.

HPLC Method

Quantification of galactooligosaccharides (GOS), lactose, glucose andgalactose were performed by HPLC. Analysis of samples was carried out ona Dionex ICS 3000. IC parameters were as follows: Mobile phase: 150 mMNaOH, Flow: Isochratic, 0.25 ml/min, Column: Carbopac PA1, Columntemperature: RT, Injection volume: 10 μL, Detector: PAD, Integration:Manual, Sample preparation: 100 times dilution in Milli-Q water (0.1 mlsample+9.9 ml water) and filtration through 0.45 ìm syringe filters,Quantification: Peak areas in percent of peak area of the standard. AGOS syrup (Vivanal GOS, Friesland Campina) was used as standard for GOSquantification. In this example, the term “GOS” is defined asgalactooligosaccharides with a degree of polymerization (DP) of 3 orabove.

Results

The quantified amount of GOS generated in the milk-base by BIF_(—)917,BIF_(—)995 and BIF_(—)1326 is shown in FIG. 4. It can be see that theshorter variants BIF_(—)917 and BIF_(—)995 have a significantly(determined by a students T-test with 95% confidence) higher GOSproduction around 1.2% (w/v) compared to BIF_(—)1326 generating below0.1% (w/v).

Example 3 Degradation Pattern of Truncation Variants

A library covering the region between BIF1230 and BIF1325 was orderedfrom GeneART (Regensburg, Germany) (see table 2). The truncationvariants was produced as described in method 1. The resulting peptideswere subjected to SDS_PAGE analysis and visualized with Simply BlueSafestain (Invitrogen, Cat# LC6060) (FIG. 5).

Results

Surprisingly, most of the variants were proteolytically modified in thefinal broth with varying amounts of target band appearing at the end offermentation. The variants generated three distinct bands with varyingintensities which was verified using mass spectrometry. The variantshave C-terminal truncation with BIF917 corresponding to the termini ofSEQ ID NO: 1, BIF995 corresponding to the termini of SEQ ID NO: 2 andBIF1068 corresponding to the termini of SEQ ID NO: 3.

The protein bands cut from the gel (marked with arrows in FIG. 5) aredigested using three different enzymes, as preparation for massspectrometry analysis. Trypsin hydrolyzes peptide bonds specifically atthe carboxyl side of arginine (R) and lysine (K) residues except when aproline (P) is on the carboxyl side. α-Chymotrypsin hydrolyzes peptidebonds specifically at the carboxyl side of tyrosine (Y), phenylalanine(F), tryptophan (W) and leucine (L) except when a proline (P) is on thecarboxyl side. Glu-C preferentially cleaves at the carboxyl side ofglutamyl (E) in ammonium bicarbonate buffer pH 8, but also cleaves atthe carboxyl side of aspartyl (D) if the hydrolysis is carried out in aphosphate buffer pH 8.

In order to detect the C-terminal, the protein of interest is preparedfor analysis using our basic procedure for protein characterisation(A2963), with one change using 40% ¹⁸O-water in the digestion buffer.The theory is that the proteolytic cleavage will incorporate both¹⁸O-water and ¹⁶O-water in the resulting peptides, which consequentlywill appear as doublets. The protein C-terminal though will only appearas a single peptide with ¹⁶O-water, since it is not cleaved but just the“last peptide” left of the protein. In this way the C-terminal is mappedusing MS/MS analysis.

TABLE 2 Variants Name Fragment of SEQ ID NO: 6 WELL BIF1230 1 1201 A01BIF1231 1 1202 B01 BIF1232 1 1203 C01 BIF1233 1 1204 BIF1234 1 1205BIF1235 1 1206 D01 BIF1236 1 1207 BIF1237 1 1208 E01 BIF1238 1 1209 F01BIF1239 1 1210 G01 BIF1240 1 1211 A02 BIF1241 1 1212 B02 BIF1242 1 1213C02 BIF1243 1 1214 BIF1244 1 1215 E02 BIF1245 1 1216 F02 BIF1246 1 1217G02 BIF1247 1 1218 H02 BIF1248 1 1219 A03 BIF1249 1 1220 B03 BIF1250 11221 C03 BIF1251 1 1222 D03 BIF1252 1 1223 E03 BIF1253 1 1224 BIF1254 11225 F03 BIF1255 1 1226 G03 BIF1256 1 1227 H03 BIF1257 1 1228 A04BIF1258 1 1229 B04 BIF1259 1 1230 C04 BIF1260 1 1231 D04 BIF1261 1 1232BIF1262 1 1233 E04 BIF1263 1 1234 F04 BIF1264 1 1235 G04 BIF1265 1 1236H04 BIF1266 1 1237 A05 BIF1267 1 1238 B05 BIF1268 1 1239 C05 BIF1269 11240 D05 BIF1270 1 1241 E05 BIF1271 1 1242 F05 BIF1272 1 1243 G05BIF1273 1 1244 H05 BIF1274 1 1245 A06 BIF1275 1 1246 B06 BIF1276 1 1247C06 BIF1277 1 1248 D06 BIF1278 1 1249 E06 BIF1279 1 1250 BIF1280 1 1251F06 BIF1281 1 1252 G06 BIF1282 1 1253 H06 BIF1283 1 1254 A07 BIF1284 11255 BIF1285 1 1256 BIF1286 1 1257 BIF1287 1 1258 B07 BIF1288 1 1259BIF1289 1 1260 C07 BIF1290 1 1261 D07 BIF1291 1 1262 BIF1292 1 1263 E07BIF1293 1 1264 BIF1294 1 1265 BIF1295 1 1266 F07 BIF1296 1 1267 G07BIF1297 1 1268 BIF1298 1 1269 H07 BIF1299 1 1270 A08 BIF1300 1 1271 B08BIF1301 1 1272 C08 BIF1302 1 1273 D08 BIF1303 1 1274 E08 BIF1304 1 1275F08 BIF1305 1 1276 G08 BIF1306 1 1277 H08 BIF1307 1 1278 A09 BIF1308 11279 B09 BIF1309 1 1280 BIF1310 1 1281 BIF1311 1 1282 BIF1312 1 1283 C09BIF1313 1 1284 BIF1314 1 1285 BIF1315 1 1286 D09 BIF1316 1 1287 E09BIF1317 1 1288 F09 BIF1318 1 1289 G09 BIF1319 1 1290 H09 BIF1320 1 1291A10 BIF1321 1 1292 B10 BIF1322 1 1293 C10 BIF1323 1 1294 D10 BIF1324 11295 E10 BIF1325 1 1296 F10

Example 4 GOS Generated Enzymatically In Situ in Milkbase and Yoghurts

In this example, the term “GOS” is defined as galactooligosaccharideswith a degree of polymerization (DP) of 3 or above.

Evaluation of GOS production by BIF917 and BIF995 were tested by in situapplication in different set-style yogurts. The β-glactosidase was addedto the milk-base simultaneous with addition of the specific yoghurtcultures, resulting in the transgalactosylation reaction runningtogether with the yoghurt fermentation process.

Initial yoghurt (set-style) batch experiments were made with a 100 mLmilkbase (yoghurt mix). The milkbase consisted of 98.60%(w/v) freshpasteurized conventional (not-organic) low-fat milk (Mini-mælk 0.5% fat,Arla Foods Amba, Denmark) and 1.4% (w/v) Nutrilac YQ-5075 wheyingredient (Arla Foods Ingredients, Denmark), resulting in a lactoseconcentration of 5.5%(w/v) corresponding to 5.3% (w/w) (1%(w/v)lactose=0.9587%(w/w) lactose in this solution). To completely hydrateNutrilac YQ-5075 the mixture was left with weak agitation for 20 hr at4° C. in the initial experiment a freeze-dried YO-MIX 485LYO culture wasused consisting of Lactobacillus delbrüeckii subsp bulgaricus andStreptococcus thermophilus (DuPont Nutrition Biosciences, Denmark). Aninitial dilution of the culture was made, adding 10 g of YO-MIX 485LYOto 400 mL UHT conventional (not-organic) milk (Let-mælk 1.5% fat, ArlaFoods Amba, Denmark). 1.43 mL of the diluted culture was added per litreof milk-base. 100 mL milkbase were distributed in 250 mL bluecap bottlesand enzymes were added in varying concentration (10, 20 and 40 ppm,corresponding to 0.213, 0.426 and 0.853 LAU as described in method 4)constitution 1% (v/v) of the final yogurt-mix. Yoghurt fermentation wasperformed at 43° C. and ended after 10 hr by fast cooling on ice.Fermentations were always run in duplicates and yoghurtsugar/oligosaccharide composition was analyzed by HPLC on the day afterfermentation (see HPLC method below). Fermented yoghurt samples werealways stored at 4° C.

The results of the initial yoghurt experiment are shown in table 3. Itcan be seen that increased dose of either BIF917 or BIF995 from 10 ppmto 40 ppm lead to increased GOS content and decreased amount of DP2(including lactose) in the final yoghurt. The difference in performanceof the two variants is within the variance of the HPLC determination andit may be concluded that they perform similar within the investigateddosages.

TABLE 3 Content of DP2 saccharides (mainly lactose) and GOS (DP3+) in afermented yogurt treated with increasing dose of BIF917 and BID995. Allresults are calculated as an average of three independent measurements.Amount Amount w/v % w/v % DP2 incl. GOS Lactose Std (DP3+) Std YogurtBIF917_10 ppm 2.191 0.092 1.249 0.051 BIF917_20 ppm 1.296 0.047 1.8820.056 BIF917_40 ppm 0.970 0.019 2.346 0.047 BIF995_10 ppm 2.787 0.1391.158 0.035 BIF995_20 ppm 1.494 0.075 1.649 0.082 BIF995_40 ppm 0.9310.028 2.392 0.063 H20 4.219 0.127 0.000 0.000 Milkbase 5.500 0.156 0.0000.000

Set-style yoghurt was made with higher initial lactose concentration of7.5% w/v (corresponding to 7.1% w/w, as 1% w/v lactose=0.9423% w/w inthis solution) to investigate its effect on the GOS concentrationachieved in the final yoghurt. The following procedure was applied:

-   -   1. All powder ingredients (listed in table 4) are mixed and the        dry blend are added to the milk/water under good agitation at        4-5° C., left to hydrate for 20 hours at 4° C.    -   2. The milkbase is preheated to 65° C. (P1)    -   3. The milkbase is homogenised at 65° C./200 bar    -   4. The milkbase is pasteurised 95° C. for 6 minutes (P3)    -   5. The milkbase is cooled to 5° C. (K2)

TABLE 4 SET yogurt ingredients list in % (w/w) Ingredients in % (w/w)Ingredient Name Skimmed milk (Skummet-mælk 0.1% fat, Arla Foods 93.533Amba, Denmark) Cream 38% fat (Arla Foods Amba, Denmark) 1.067 NutrilacYQ5075 (Arla Foods Ingredients, Denmark) 1.400 Lactose (Variolac ® 992BG100, Arla Foods Amba, Denmark) 3.000 Enzyme/H20 1.000 Total % 100

Following, the milkbase is heated to 43° C. (K1). Dilution of theculture YO-MIX 495 consisting of Lactobacillus delbrüeckii subspbulgaricus and Streptococcus thermophilus (DuPont Nutrition Biosciences,Denmark) was done at the fermentation temperature. 250 mL milkbase weredistributed in 500 mL bluecap bottles and enzymes were added in varyingconcentration constitution 1% (v/v) of the final yogurt-mix The starterculture, YO-Mix 495, are added in a dosage of 20 DCU (Danisco CultureUnits) per 100 litre, where one DCU is 100 billion cells measured ascolony forming units. For each trial three samples were made.Fermentation was carried out to pH 4.6 at 43° C. and following stoppedby fast cooling to 5° C. Yoghurt sugar/oligosaccharide composition wasanalyzed by HPLC on the day after fermentation (see HPLC method below).Fermented yoghurt samples were always stored at 4° C.

The results of the yoghurt experiment are shown in table 5. It can beseen that higher initial lactose (7.5% w/v) increased the total GOSgenerated in the yoghurt compared to the GOS achieved with 5.5% (w/v)initial lactose, as shown in table 3. A final GOS concentration of2.954% is achieved with the 50 ppm dose of BIF917 tested, whereas 2.662%GOS was produced with 25 ppm BIF917. In comparison did the conventionalhydrolyzing β-galactosidase from Kluyveromyces lactis (GODO-YNL2, GODOSHUSEI Co., Ltd., Japan) produced 0.355% GOS at a dose of 25 ppm. Adecrease in the lactose concentration of 2.127% was observed in theblank yogurt where the same amount of H₂O was added instead of enzyme.

TABLE 5 Content of DP2 saccharides (mainly lactose) and GOS (DP3+) in afermented yogurt treated with increasing dose of BIF917 and K. lactisβ-gal. Amount Amount w/v % w/v % DP2 incl. DP3+ Lactose Std (GOS) StdYogurt BIF917_12.5 ppm 3.295 0.224 1.525 0.197 BIF917_25 ppm 2.090 0.0452.662 0.003 BIF917_50 ppm 1.395 0.090 2.954 0.202 K. lactis β-Gal_25 ppm0.425 0.040 0.355 0.006 H20 5.431 0.099 0.000 0.000 Milkbase H20 7.5580.265 0.000 0.000

To test the influence of acidification in the yogurt fermentation onBIF917 performance studies were made with three different YO-mixcultures all consisting of Lactobacillus delbrüeckii subsp bulgaricusand Streptococcus thermophilus (DuPont Nutrition Biosciences, Denmark):YO-MIX 495 with a relative slow fermentation time; YO-MIX 495 with arelative fast fermentation time and YO-MIX 601 with prolonged lagphaseand a strong acidifying fermentation. All fermentations were performedat 43° C. with the same amount of BIF917 (25 ppm).

In addition to test the influence of temperature on BIF917 performance,fermentation with YO-MIX 495 and 25 ppm BIF917 were carried out at 43°C., 45° C. and 47° C.

The following procedure was applied to produce set-style yogurts with aninitial lactose concentration of 7.5% (w/v) (corresponding to 7.1% w/w,as 1% w/v lactose=0.9423% w/w in this solution):

-   -   1. All powder ingredients (listed in table 6) are mixed and the        dry blend are added to the milk/water under good agitation at        4-5° C., left to hydrate for 20 hours at 4° C.    -   2. The milkbase is preheated to 65° C. (P1)    -   3. The milkbase is homogenised at 65° C./200 bar    -   4. The milkbase is pasteurised 95° C. for 6 minutes (P3)    -   5. The milkbase is cooled to 5° C. (K2)    -   6. The milkbase is heated to 43° C. (K1). Dilution of the        cultures YO-MIX 495, 485 and 601 (DuPont Nutrition Biosciences,        Denmark) was done at the fermentation temperature.    -   7. 100 mL milkbase was distributed in 250 mL bluecap bottles and        enzymes were added in varying concentration constitution 1%        (v/v) of the final yogurt-mix    -   8. The starter culture, either YO-Mix 495, 485 or 601 were added        in a dosage of 20 DCU (Danisco Culture Units) per 100 litre. For        each trial three samples were made.    -   9. Fermentation was carried out to pH 4.6 at 43° C. (for        temperature studies at 43° C., 45° C. and 47° C. respectively)        and following stopped by fast cooling to 5° C.    -   10. Yoghurt sugar/oligosaccharide composition was analyzed by        HPLC on the day after fermentation (see HPLC method below).        Fermented yoghurt samples were always stored at 4° C.

TABLE 6 SET yogurt ingredients list in % (w/w) Ingredients in % (w/w)Ingredient Name Skimmed milk (Skummet-mælk 0.1% fat, Arla Foods 93.533Amba, Denmark) Cream 38% fat (Arla Foods Amba, Denmark) 1.067 NutrilacYQ5215 (Arla Foods Ingredients, Denmark) 1.400 Lactose (Variolac ® 992BG100, Arla Foods Amba, Denmark) 3.000 Enzyme/H20 1.000 Total % 100

The results of the yoghurt experiment are shown in table 7. It can beseen that the different YO-mix cultures, having different acidificationprofiles, exert no significant effect on the final GOS yield. On average3.22% (w/v) GOS is generated and the highest GOS concentration (3.300%)found in the yoghurt produced with YO-mix 485 is being within thevariance of quantification. The change in fermentation temperature from43° C. to 45° C. and 47° C. do not significantly (using a student T-testwith 95% confidence limits) change the amount of GOS produced in any ofthe yoghurts: 3.258% w/v at 43° C., 3.375% w/v at 45° C. and 3.236% w/vat 47° C. Thus, it may be concluded that the action of BIF917 under theconditions investigated are robust for in situ generation of GOS inyogurt using various culture and temperature conditions.

TABLE 7 Content of DP2, DP3, DP4, DP5, DP6, glucose and galactose in afermented yogurt treated with BIF917. Fermen- Fermen- tation LactoseGlucose Galactose DP3 (GOS) DP4 (GOS) DP5 (GOS) DP6 (GOS) DP3+ (GOS)tation temper- w/v % w/v % w/v % w/v % w/v % w/v % w/v % w/v % SampleEnzyme dose culture ature Amount Amount Amount Amount Amount AmountAmount Amount Milkbase 7.259 0.000 0.000 0.000 0.000 0.000 0.000 0.000Yogurt BIF917_25 ppm YM 495 43° C. 5.466 0.000 0.448 0.000 0.000 0.0000.000 0.000 BIF917_25 ppm YM 495 43° C. 1.770 1.368 0.648 2.067 0.7930.260 0.074 3.194 BIF917_25 ppm YM 485 43° C. 1.860 1.259 0.609 2.0650.830 0.302 0.103 3.300 BIF917_25 ppm YM 601 43° C. 1.712 1.418 0.8322.094 0.780 0.248 0.068 3.189 BIF917_25 ppm YM 495 43° C. 1.761 1.3440.642 2.086 0.813 0.275 0.084 3.258 BIF917_25 ppm YM 495 45° C. 1.8381.323 0.625 2.128 0.848 0.301 0.099 3.375 BIF917_25 ppm YM 495 47° C.1.739 1.406 0.722 2.106 0.799 0.257 0.074 3.236 STD Milkbase 0.252 0.0000.000 0.000 0.000 0.000 0.000 0.000 Yogurt BIF917_25 ppm YM 495 43° C.0.225 0.000 0.000 0.000 0.000 0.000 0.000 0.000 BIF917_25 ppm YM 495 43°C. 0.075 0.067 0.039 0.080 0.031 0.010 0.002 0.123 BIF917_25 ppm YM 48543° C. 0.090 0.063 0.015 0.084 0.033 0.012 0.008 0.136 BIF917_25 ppm YM601 43° C. 0.013 0.014 0.017 0.018 0.010 0.006 0.002 0.037 BIF917_25 ppmYM 495 43° C. 0.042 0.031 0.014 0.046 0.016 0.005 0.001 0.067 BIF917_25ppm YM 495 45° C. 0.071 0.050 0.013 0.061 0.028 0.012 0.007 0.108BIF917_25 ppm YM 495 47° C. 0.009 0.015 0.004 0.003 0.002 0.003 0.0030.011

HPLC Method

All chemicals used were of analytical grade. D-(+)-Lactose (min 99%, no.17814), D-(+)-glucose (min 99.5%, no G8270-100G, batch#036K0137), andD-(+)-galactose (min 99%, no G0750-25G, batch#031M0043V) were obtainedfrom Sigma (St. Louis, Mo., USA). Vivinal GOS Syrup (Prod. No 502675Batch#649566) was obtained from Friesland Campina Domo (Amersfoort, TheNetherlands) containing 57% on dry-matter (DM) galacto-oligosaccharides,21% on DM anhydrous lactose, 20% on DM anhydrous glucose, and 0.8% on DManhydrous galactose.

Sample Preparation

All standards: Lactose, Glucose, galactose and GOS were prepared indouble distilled water (ddH20) and filtered through 0.45 μm syringefilters. A set of each standard was prepared ranging in concentrationfrom 10 to 200000 ppm.

To evaluate quantification of the above set of sugars in a yogurt/milkmatrix, the above standards were spiked into a milk and yogurt sample asinternal controls. All milk and yogurt samples containing activeβ-galactosidase were inactivated by heating the sample to 95° C. for 10min. All milk samples were prepared in 96 well MTP plates (Corning,N.Y., USA) and diluted minimum 20 times and filtered through 0.20 μm 96well plate filters before analysis (Corning filter plate, PVDFhydrophile membrane, NY, USA). Samples containing more than 50000 ppm(5% w/v) lactose were heated to 30° C. to ensure proper solubilization.All yogurt samples were weighted and diluted 10 times in ddH20 beforehomogenization of the sample using of Ultra turrax TP18/10 for a fewminutes (Janke & Kunkel Ika-labortechnik, Bie & Berntsen, Denmark).β-galactosidase were inactivated by heat treatment and samples werefurther diluted in 96 well MTP plates filtered through 0.20 μm 96 wellplate filters before analysis (Corning filter plate, PVDF hydrophilemembrane, NY, USA). All samples were analyzed in 96 well MTP platessealed with tape.

Instrumentation

Quantification of galactooligosaccharides (GOS), Lactose, glucose andgalactose were performed by HPLC. Analysis of samples was carried out ona Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific) equippedwith a DGP-3600SD Dual-Gradient analytical pump, WPS-3000TSLthermostated autosampler, TCC-3000SD thermostated column oven, and aRI-101 refractive index detector (Shodex, J M Science). Chromeleondatasystem software (Version 6.80, DU10A Build 2826, 171948) was usedfor data acquisition and analysis.

Chromatographic Conditions

The samples were analyzed by HPLC using a RSO oligosaccharide column,Ag⁺ 4% crosslinked (Phenomenex, The Netherlands) equipped with ananalytical guard column (Carbo-Ag⁺ neutral, AJ0-4491, Phenomenex, TheNetherlands) at 70° C. The column was eluted with double distilled water(filtered through a regenerated cellulose membrane of 0.45 μm and purgedwith helium gas) at a flow rate of 0.3 ml/min.

Isocratic flow of 0.3 ml/min was maintained throughout analysis with atotal run time of 37 min and injection volume was set to 10 μL. Sampleswere held at 30° C. in the thermostated autosampler compartment toensure solubilisation of lactose. The eluent was monitored by means of arefractive index detector and quantification was made by the peak arearelative to the peak area of the given standard. Peaks with a degree ofthree or higher (DP3+) in the Vivinal GOS syrup (Friesland Food Domo,The Netherlands) were used as standard for GOS quantification followingmanufactures declaration on GOS content in the product.

LIST OF SEQUENCES >SEQ ID NO: 1 (BIF_917)vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwkfmlsdsvqaqdpafddsawqqvdlphdysitqkysqsneaesaylpggtgwyrksftidrdlagkriainfdgvymnatvwfngvklgthpygyspfsfdltgnakfggentivvkvenrlpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslatqnggdvtmnlttkvandteaaanitlkqtvfpkggktdaaigtvttasksiaagasadvtstitaaspklwsiknpnlytvrtevlnggkvldtydteygfrwtgfdatsgfslngekvklkgvsmhhdqgslgavanrraierqveilqkmgvnsirtthnpaakalidvcnekgvlvveevfdmwnrskngntedygkwfgqaiagdnavlggdkdetwakfdltstinrdrnapsvimwslgnemmegisgsvsgfpatsaklvawtkaadstrpmtygdnkikanwnesntmgdnltanggvvgtnysdganydkirtthpswaiygsetasainsrgiynrttggaqssdkqltsydnsavgwgavassawydvvqrdfvagtyvwtgfdylgeptpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqsqwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvklyftpkgstekrligeksftkkttaagytyqvyegsdkdstahknmyltwnvpwaegtisaeaydennrlipegstegnasvtttgkaaklkadadrktitadgkdlsyievdvtdanghivpdaanrvtfdvkgagklvgvdngsspdhdsyqadnrkafsgkvlaivqstkeageitvtakadglqsstvkiattavpgtstekt >SEQ ID NO: 2 (BIF_995)vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwkfmlsdsvqaqdpafddsawqqvdlphdysitqkysqsneaesaylpggtgwyrksftidrdlagkriainfdgvymnatvwfngvklgthpygyspfsfdltgnakfggentivvkvenrlpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslatqnggdvtmnlttkvandteaaanitlkqtvfpkggktdaaigtvttasksiaagasadvtstitaaspklwsiknpnlytvrtevlnggkvldtydteygfrwtgfdatsgfslngekvklkgvsmhhdqgslgavanrraierqveilqkmgvnsirtthnpaakalidvcnekgvlvveevfdmwnrskngntedygkwfgqaiagdnavlggdkdetwakfdltstinrdrnapsvimwslgnemmegisgsvsgfpatsaklvawtkaadstrpmtygdnkikanwnesntmgdnltanggvvgtnysdganydkirtthpswaiygsetasainsrgiynrttggaqssdkqltsydnsavgwgavassawydvvqrdfvagtyvwtgfdylgeptpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqsqwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvklyftpkgstekrligeksftkkttaagytyqvyegsdkdstahknmyltwnvpwaegtisaeaydennrlipegstegnasvtttgkaaklkadadrktitadgkdlsyievdvtdanghivpdaanrvtfdvkgagklvgvdngsspdhdsyqadnrkafsgkvlaivqstkeageitvtakadglqsstvkiattavpgtstektvrsfyysrnyyvktgnkpilpsdvevrysdgtsdrqnvtwdavsddqiakagsfsvagtvagqkisvrvtmideigal >SEQ ID NO: 3 (BIF_1068)Vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwkfmlsdsvqaqdpafddsawqqvdlphdysitqkysqsneaesaylpggtgwyrksftidrdlagkriainfdgvymnatvwfngvklgthpygyspfsfdltgnakfggentivvkvenrlpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslatqnggdvtmnlttkvandteaaanitlkqtvfpkggktdaaigtvttasksiaagasadvtstitaaspklwsiknpnlytvrtevlnggkvldtydteygfrwtgfdatsgfslngekvklkgvsmhhdqgslgavanrraierqveilqkmgvnsirtthnpaakalidvcnekgvlvveevfdmwnrskngntedygkwfgqaiagdnavlggdkdetwakfdltstinrdrnapsvimwslgnemmegisgsvsgfpatsaklvawtkaadstrpmtygdnkikanwnesntmgdnltanggvvgtnysdganydkirtthpswaiygsetasainsrgiynrttggaqssdkqltsydnsavgwgavassawydvvqrdfvagtyvwtgfdylgeptpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqsqwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvklyftpkgstekrligeksftkkttaagytyqvyegsdkdstahknmyltwnvpwaegtisaeaydennrlipegstegnasvtttgkaaklkadadrktitadgkdlsyievdvtdanghivpdaanrvtfdvkgagklvgvdngsspdhdsyqadnrkafsgkvlaivqstkeageitvtakadglqsstvkiattavpgtstektvrsfyysrnyyvktgnkpilpsdvevrysdgtsdrqnvtwdavsddqiakagsfsvagtvagqkisvrvtmideigallnysastpvgtpavlpgsrpavlpdgtvtsanfavhwtkpadtvyntagtvkvpgtatvfgkefkvtatirvq >SEQ ID NO: 4 (BIF_1172)vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwkfmlsdsvqaqdpafddsawqqvdlphdysitqkysqsneaesaylpggtgwyrksftidrdlagkriainfdgvymnatvwfngvklgthpygyspfsfdltgnakfggentivvkvenrlpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslatqnggdvtmnlttkvandteaaanitlkqtvfpkggktdaaigtvttasksiaagasadvtstitaaspklwsiknpnlytvrtevlnggkvldtydteygfrwtgfdatsgfslngekvklkgvsmhhdqgslgavanrraierqveilqkmgvnsirtthnpaakalidvcnekgvlvveevfdmwnrskngntedygkwfgqaiagdnavlggdkdetwakfdltstinrdrnapsvimwslgnemmegisgsvsgfpatsaklvawtkaadstrpmtygdnkikanwnesntmgdnltanggvvgtnysdganydkirtthpswaiygsetasainsrgiynrttggaqssdkqltsydnsavgwgavassawydvvqrdfvagtyvwtgfdylgeptpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqsqwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvklyftpkgstekrligeksftkkttaagytyqvyegsdkdstahknmyltwnvpwaegtisaeaydennrlipegstegnasvtttgkaaklkadadrktitadgkdlsyievdvtdanghivpdaanrvtfdvkgagklvgvdngsspdhdsyqadnrkafsgkvlaivqstkeageitvtakadglqsstvkiattavpgtstektvrsfyysrnyyvktgnkpilpsdvevrysdgtsdrqnvtwdavsddqiakagsfsvagtvagqkisvrvtmideigallnysastpvgtpavlpgsrpavlpdgtvtsanfavhwtkpadtvyntagtvkvpgtatvfgkefkvtatirvqrsqvtigssvsgnalrltqnipadkqsdtldaikdgsttvdantggganpsawtnwayskaghntaeitfeyateqqlgqivmyffrdsnavrfpdagktkiqi >SEQ ID NO: 5 (BIF_1241)vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwkfmlsdsvqaqdpafddsawqqvdlphdysitqkysqsneaesaylpggtgwyrksftidrdlagkriainfdgvymnatvwfngvklgthpygyspfsfdltgnakfggentivvkvenrlpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslatqnggdvtmnlttkvandteaaanitlkqtvfpkggktdaaigtvttasksiaagasadvtstitaaspklwsiknpnlytvrtevlnggkvldtydteygfrwtgfdatsgfslngekvklkgvsmhhdqgslgavanrraierqveilqkmgvnsirtthnpaakalidvcnekgvlvveevfdmwnrskngntedygkwfgqaiagdnavlggdkdetwakfdltstinrdrnapsvimwslgnemmegisgsvsgfpatsaklvawtkaadstrpmtygdnkikanwnesntmgdnltanggvvgtnysdganydkirtthpswaiygsetasainsrgiynrttggaqssdkqltsydnsavgwgavassawydvvqrdfvagtyvwtgfdylgeptpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqsqwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvklyftpkgstekrligeksftkkttaagytyqvyegsdkdstahknmyltwnvpwaegtisaeaydennrlipegstegnasvtttgkaaklkadadrktitadgkdlsyievdvtdanghivpdaanrvtfdvkgagklvgvdngsspdhdsyqadnrkafsgkvlaivqstkeageitvtakadglqsstvkiattavpgtstektvrsfyysrnyyvktgnkpilpsdvevrysdgtsdrqnvtwdavsddqiakagsfsvagtvagqkisvrvtmideigallnysastpvgtpavlpgsrpavlpdgtvtsanfavhwtkpadtvyntagtvkvpgtatvfgkefkvtatirvqrsqvtigssvsgnalrltqnipadkqsdtldaikdgsttvdantggganpsawtnwayskaghntaeitfeyateqqlgqivmyffrdsnavrfpdagktkiqisadgknwtdlaatetiaaqessdrvkpytydfapvgatfvkvtvtnadtttpsgvvcaglteielkt >SEQID NO: 6 (BIF_1326)vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwkfmlsdsvqaqdpafddsawqqvdlphdysitqkysqsneaesaylpggtgwyrksftidrdlagkriainfdgvymnatvwfngvklgthpygyspfsfdltgnakfggentivvkvenrlpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslatqnggdvtmnlttkvandteaaanitlkqtvfpkggktdaaigtvttasksiaagasadvtstitaaspklwsiknpnlytvrtevlnggkvldtydteygfrwtgfdatsgfslngekvklkgvsmhhdqgslgavanrraierqveilqkmgvnsirtthnpaakalidvcnekgvlvveevfdmwnrskngntedygkwfgqaiagdnavlggdkdetwakfdltstinrdrnapsvimwslgnemmegisgsvsgfpatsaklvawtkaadstrpmtygdnkikanwnesntmgdnltanggvvgtnysdganydkirtthpswaiygsetasainsrgiynrttggaqssdkqltsydnsavgwgavassawydvvqrdfvagtyvwtgfdylgeptpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqsqwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvklyftpkgstekrligeksftkkttaagytyqvyegsdkdstahknmyltwnvpwaegtisaeaydennrlipegstegnasvtttgkaaklkadadrktitadgkdlsyievdvtdanghivpdaanrvtfdvkgagklvgvdngsspdhdsyqadnrkafsgkvlaivqstkeageitvtakadglqsstvkiattavpgtstektvrsfyysrnyyvktgnkpilpsdvevrysdgtsdrqnvtwdavsddqiakagsfsvagtvagqkisvrvtmideigallnysastpvgtpavlpgsrpavlpdgtvtsanfavhwtkpadtvyntagtvkvpgtatvfgkefkvtatirvqrsqvtigssvsgnalrltqnipadkqsdtldaikdgsttvdantggganpsawtnwayskaghntaeitfeyateqqlgqivmyffrdsnavrfpdagktkiqisadgknwtdlaatetiaaqessdrvkpytydfapvgatfvkvtvtnadtttpsgvvcaglteielktatskfvtntsaalssltvngtkvsdsvlaagsyntpaiiadvkaegegnasvtvlpahdnvirvitesedhvtrktftinlgteqef >SEQID NO: 7 Bifidobacterium bifidum glycoside hydrolase catalytic coreqnrtsdfdanwkfmlsdsvqaqdpafddsawqqvdlphdysitqkysqsneaesaylpggtgwyrksftidrdlagkriainfdgvymnatvwfngvklgthpygyspfsfdltgnakfggentivvkvenrlpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslatqnggdvtmnlttkvandteaaanitlkqtvfpkggktdaaigtvttasksiaagasadvtstitaaspklwsiknpnlytvrtevlnggkvldtydteygfrwtgfdatsgfslngekvklkgvsmhhdqgslgavanrraierqveilqkmgvnsirtthnpaakalidvcnekgvlvveevfdmwnrskngntedygkwfgqaiagdnavlggdkdetwakfdltstinrdmapsvimwslgnemmegisgsvsgfpatsaklvawtkaadstrpmty >SEQ ID NO: 8 nucleotide sequence encoding fulllengthgcagttgaagatgcaacaagaagcgatagcacaacacaaatgtcatcaacaccggaagttgtttattcatcagcggtcgatagcaaacaaaatcgcacaagcgattttgatgcgaactggaaatttatgctgtcagatagcgttcaagcacaagatccggcatttgatgattcagcatggcaacaagttgatctgccgcatgattatagcatcacacagaaatatagccaaagcaatgaagcagaatcagcatatcttccgggaggcacaggctggtatagaaaaagctttacaattgatagagatctggcaggcaaacgcattgcgattaattttgatggcgtctatatgaatgcaacagtctggtttaatggcgttaaactgggcacacatccgtatggctattcaccgttttcatttgatctgacaggcaatgcaaaatttggcggagaaaacacaattgtcgtcaaagttgaaaatagactgccgtcatcaagatggtattcaggcagcggcatttatagagatgttacactgacagttacagatggcgttcatgttggcaataatggcgtcgcaattaaaacaccgtcactggcaacacaaaatggcggagatgtcacaatgaacctgacaacaaaagtcgcgaatgatacagaagcagcagcgaacattacactgaaacagacagtttttccgaaaggcggaaaaacggatgcagcaattggcacagttacaacagcatcaaaatcaattgcagcaggcgcatcagcagatgttacaagcacaattacagcagcaagcccgaaactgtggtcaattaaaaacccgaacctgtatacagttagaacagaagttctgaacggaggcaaagttctggatacatatgatacagaatatggctttcgctggacaggctttgatgcaacatcaggcttttcactgaatggcgaaaaagtcaaactgaaaggcgttagcatgcatcatgatcaaggctcacttggcgcagttgcaaatagacgcgcaattgaaagacaagtcgaaatcctgcaaaaaatgggcgtcaatagcattcgcacaacacataatccggcagcaaaagcactgattgatgtctgcaatgaaaaaggcgttctggttgtcgaagaagtctttgatatgtggaaccgcagcaaaaatggcaacacggaagattatggcaaatggtttggccaagcaattgcaggcgataatgcagttctgggaggcgataaagatgaaacatgggcgaaatttgatcttacatcaacaattaaccgcgatagaaatgcaccgtcagttattatgtggtcactgggcaatgaaatgatggaaggcatttcaggctcagtttcaggctttccggcaacatcagcaaaactggttgcatggacaaaagcagcagattcaacaagaccgatgacatatggcgataacaaaattaaagcgaactggaacgaatcaaatacaatgggcgataatctgacagcaaatggcggagttgttggcacaaattattcagatggcgcaaactatgataaaattcgtacaacacatccgtcatgggcaatttatggctcagaaacagcatcagcgattaatagccgtggcatttataatagaacaacaggcggagcacaatcatcagataaacagctgacaagctatgataattcagcagttggctggggagcagttgcatcatcagcatggtatgatgttgttcagagagattttgtcgcaggcacatatgtttggacaggatttgattatctgggcgaaccgacaccgtggaatggcacaggctcaggcgcagttggctcatggccgtcaccgaaaaatagctattttggcatcgttgatacagcaggctttccgaaagatacatattatttttatcagagccagtggaatgatgatgttcatacactgcatattcttccggcatggaatgaaaatgttgttgcaaaaggctcaggcaataatgttccggttgtcgtttatacagatgcagcgaaagtgaaactgtattttacaccgaaaggctcaacagaaaaaagactgatcggcgaaaaatcatttacaaaaaaaacaacagcggcaggctatacatatcaagtctatgaaggcagcgataaagattcaacagcgcataaaaacatgtatctgacatggaatgttccgtgggcagaaggcacaatttcagcggaagcgtatgatgaaaataatcgcctgattccggaaggcagcacagaaggcaacgcatcagttacaacaacaggcaaagcagcaaaactgaaagcagatgcggatcgcaaaacaattacagcggatggcaaagatctgtcatatattgaagtcgatgtcacagatgcaaatggccatattgttccggatgcagcaaatagagtcacatttgatgttaaaggcgcaggcaaactggttggcgttgataatggctcatcaccggatcatgattcatatcaagcggataaccgcaaagcattttcaggcaaagtcctggcaattgttcagtcaacaaaagaagcaggcgaaattacagttacagcaaaagcagatggcctgcaatcaagcacagttaaaattgcaacaacagcagttccgggaacaagcacagaaaaaacagtccgcagcttttattacagccgcaactattatgtcaaaacaggcaacaaaccgattctgccgtcagatgttgaagttcgctattcagatggaacaagcgatagacaaaacgttacatgggatgcagtttcagatgatcaaattgcaaaagcaggctcattttcagttgcaggcacagttgcaggccaaaaaattagcgttcgcgtcacaatgattgatgaaattggcgcactgctgaattattcagcaagcacaccggttggcacaccggcagttcttccgggatcaagaccggcagtcctgccggatggcacagtcacatcagcaaattttgcagtccattggacaaaaccggcagatacagtctataatacagcaggcacagtcaaagtaccgggaacagcaacagtttttggcaaagaatttaaagtcacagcgacaattagagttcaaagaagccaagttacaattggctcatcagtttcaggaaatgcactgagactgacacaaaatattccggcagataaacaatcagatacactggatgcgattaaagatggctcaacaacagttgatgcaaatacaggcggaggcgcaaatccgtcagcatggacaaattgggcatattcaaaagcaggccataacacagcggaaattacatttgaatatgcgacagaacaacaactgggccagatcgtcatgtatttttttcgcgatagcaatgcagttagatttccggatgctggcaaaacaaaaattcagatcagcgcagatggcaaaaattggacagatctggcagcaacagaaacaattgcagcgcaagaatcaagcgatagagtcaaaccgtatacatatgattttgcaccggttggcgcaacatttgttaaagtgacagtcacaaacgcagatacaacaacaccgtcaggcgttgtttgcgcaggcctgacagaaattgaactgaaaacagcgacaagcaaatttgtcacaaatacatcagcagcactgtcatcacttacagtcaatggcacaaaagtttcagattcagttctggcagcaggctcatataacacaccggcaattatcgcagatgttaaagcggaaggcgaaggcaatgcaagcgttacagtccttccggcacatgataatgttattcgcgtcattacagaaagcgaagatcatgtcacacgcaaaacatttacaatcaacctgggcacagaacaagaatttccggctgattcagatgaaagagattatccggcagcagatatgacagtcacagttggctcagaacaaacatcaggcacagcaacagaaggaccgaaaaaatttgcagtcgatggcaacacatcaacatattggcatagcaattggacaccgacaacagttaatgatctgtggatcgcgtttgaactgcaaaaaccgacaaaactggatgcactgagatatcttccgcgtccggcaggctcaaaaaatggcagcgtcacagaatataaagttcaggtgtcagatgatggaacaaactggacagatgcaggctcaggcacatggacaacggattatggctggaaactggcggaatttaatcaaccggtcacaacaaaacatgttagactgaaagcggttcatacatatgcagatagcggcaacgataaatttatgagcgcaagcgaaattagactgagaaaagcggtcgatacaacggatatttcaggcgcaacagttacagttccggcaaaactgacagttgatagagttgatgcagatcatccggcaacatttgcaacaaaagatgtcacagttacactgggagatgcaacactgagatatggcgttgattatctgctggattatgcaggcaatacagcagttggcaaagcaacagtgacagttagaggcattgataaatattcaggcacagtcgcgaaaacatttacaattgaactgaaaaatgcaccggcaccggaaccgacactgacatcagttagcgtcaaaacaaaaccgagcaaactgacatatgttgtcggagatgcatttgatccggcaggcctggttctgcaacatgatagacaagcagatagacctccgcaaccgctggttggcgaacaagcggatgaacgcggactgacatgcggcacaagatgcgatagagttgaacaactgcgcaaacatgaaaatagagaagcgcatagaacaggcctggatcatctggaatttgttggcgcagcagatggcgcagttggagaacaagcaacatttaaagtccatgtccatgcagatcagggagatggcagacatgatgatgcagatgaacgcgatattgatccgcatgttccggtcgatcatgcagttggcgaactggcaagagcagcatgccatcatgttattggcctgagagtcgatacacatagacttaaagcaagcggctttcaaattccggctgatgatatggcagaaatcgatcgcattacaggctttcatcgttttgaacgccatgtc >SEQ ID NO: 9nucleotide sequence encoding BIF_917gttgaagatgcaacaagaagcgatagcacaacacaaatgtcatcaacaccggaagttgtttattcatcagcggtcgatagcaaacaaaatcgcacaagcgattttgatgcgaactggaaatttatgctgtcagatagcgttcaagcacaagatccggcatttgatgattcagcatggcaacaagttgatctgccgcatgattatagcatcacacagaaatatagccaaagcaatgaagcagaatcagcatatcttccgggaggcacaggctggtatagaaaaagctttacaattgatagagatctggcaggcaaacgcattgcgattaattttgatggcgtctatatgaatgcaacagtctggtttaatggcgttaaactgggcacacatccgtatggctattcaccgttttcatttgatctgacaggcaatgcaaaatttggcggagaaaacacaattgtcgtcaaagttgaaaatagactgccgtcatcaagatggtattcaggcagcggcatttatagagatgttacactgacagttacagatggcgttcatgttggcaataatggcgtcgcaattaaaacaccgtcactggcaacacaaaatggcggagatgtcacaatgaacctgacaacaaaagtcgcgaatgatacagaagcagcagcgaacattacactgaaacagacagtttttccgaaaggcggaaaaacggatgcagcaattggcacagttacaacagcatcaaaatcaattgcagcaggcgcatcagcagatgttacaagcacaattacagcagcaagcccgaaactgtggtcaattaaaaacccgaacctgtatacagttagaacagaagttctgaacggaggcaaagttctggatacatatgatacagaatatggctttcgctggacaggctttgatgcaacatcaggcttttcactgaatggcgaaaaagtcaaactgaaaggcgttagcatgcatcatgatcaaggctcacttggcgcagttgcaaatagacgcgcaattgaaagacaagtcgaaatcctgcaaaaaatgggcgtcaatagcattcgcacaacacataatccggcagcaaaagcactgattgatgtctgcaatgaaaaaggcgttctggttgtcgaagaagtctttgatatgtggaaccgcagcaaaaatggcaacacggaagattatggcaaatggtttggccaagcaattgcaggcgataatgcagttctgggaggcgataaagatgaaacatgggcgaaatttgatcttacatcaacaattaaccgcgatagaaatgcaccgtcagttattatgtggtcactgggcaatgaaatgatggaaggcatttcaggctcagtttcaggctttccggcaacatcagcaaaactggttgcatggacaaaagcagcagattcaacaagaccgatgacatatggcgataacaaaattaaagcgaactggaacgaatcaaatacaatgggcgataatctgacagcaaatggcggagttgttggcacaaattattcagatggcgcaaactatgataaaattcgtacaacacatccgtcatgggcaatttatggctcagaaacagcatcagcgattaatagccgtggcatttataatagaacaacaggcggagcacaatcatcagataaacagctgacaagctatgataattcagcagttggctggggagcagttgcatcatcagcatggtatgatgttgttcagagagattttgtcgcaggcacatatgtttggacaggatttgattatctgggcgaaccgacaccgtggaatggcacaggctcaggcgcagttggctcatggccgtcaccgaaaaatagctattttggcatcgttgatacagcaggctttccgaaagatacatattatttttatcagagccagtggaatgatgatgttcatacactgcatattcttccggcatggaatgaaaatgttgttgcaaaaggctcaggcaataatgttccggttgtcgtttatacagatgcagcgaaagtgaaactgtattttacaccgaaaggctcaacagaaaaaagactgatcggcgaaaaatcatttacaaaaaaaacaacagcggcaggctatacatatcaagtctatgaaggcagcgataaagattcaacagcgcataaaaacatgtatctgacatggaatgttccgtgggcagaaggcacaatttcagcggaagcgtatgatgaaaataatcgcctgattccggaaggcagcacagaaggcaacgcatcagttacaacaacaggcaaagcagcaaaactgaaagcagatgcggatcgcaaaacaattacagcggatggcaaagatctgtcatatattgaagtcgatgtcacagatgcaaatggccatattgttccggatgcagcaaatagagtcacatttgatgttaaaggcgcaggcaaactggttggcgttgataatggctcatcaccggatcatgattcatatcaagcggataaccgcaaagcattttcaggcaaagtcctggcaattgttcagtcaacaaaagaagcaggcgaaattacagttacagcaaaagcagatggcctgcaatcaagcacagttaaaattgcaacaacagcagttccgggaacaagcacagaaaaaaca >SEQ ID NO:10 nucleotide sequence encoding BIF_995gttgaagatgcaacaagaagcgatagcacaacacaaatgtcatcaacaccggaagttgtttattcatcagcggtcgatagcaaacaaaatcgcacaagcgattttgatgcgaactggaaatttatgctgtcagatagcgttcaagcacaagatccggcatttgatgattcagcatggcaacaagttgatctgccgcatgattatagcatcacacagaaatatagccaaagcaatgaagcagaatcagcatatcttccgggaggcacaggctggtatagaaaaagctttacaattgatagagatctggcaggcaaacgcattgcgattaattttgatggcgtctatatgaatgcaacagtctggtttaatggcgttaaactgggcacacatccgtatggctattcaccgttttcatttgatctgacaggcaatgcaaaatttggcggagaaaacacaattgtcgtcaaagttgaaaatagactgccgtcatcaagatggtattcaggcagcggcatttatagagatgttacactgacagttacagatggcgttcatgttggcaataatggcgtcgcaattaaaacaccgtcactggcaacacaaaatggcggagatgtcacaatgaacctgacaacaaaagtcgcgaatgatacagaagcagcagcgaacattacactgaaacagacagtttttccgaaaggcggaaaaacggatgcagcaattggcacagttacaacagcatcaaaatcaattgcagcaggcgcatcagcagatgttacaagcacaattacagcagcaagcccgaaactgtggtcaattaaaaacccgaacctgtatacagttagaacagaagttctgaacggaggcaaagttctggatacatatgatacagaatatggctttcgctggacaggctttgatgcaacatcaggcttttcactgaatggcgaaaaagtcaaactgaaaggcgttagcatgcatcatgatcaaggctcacttggcgcagttgcaaatagacgcgcaattgaaagacaagtcgaaatcctgcaaaaaatgggcgtcaatagcattcgcacaacacataatccggcagcaaaagcactgattgatgtctgcaatgaaaaaggcgttctggttgtcgaagaagtctttgatatgtggaaccgcagcaaaaatggcaacacggaagattatggcaaatggtttggccaagcaattgcaggcgataatgcagttctgggaggcgataaagatgaaacatgggcgaaatttgatcttacatcaacaattaaccgcgatagaaatgcaccgtcagttattatgtggtcactgggcaatgaaatgatggaaggcatttcaggctcagtttcaggctttccggcaacatcagcaaaactggttgcatggacaaaagcagcagattcaacaagaccgatgacatatggcgataacaaaattaaagcgaactggaacgaatcaaatacaatgggcgataatctgacagcaaatggcggagttgttggcacaaattattcagatggcgcaaactatgataaaattcgtacaacacatccgtcatgggcaatttatggctcagaaacagcatcagcgattaatagccgtggcatttataatagaacaacaggcggagcacaatcatcagataaacagctgacaagctatgataattcagcagttggctggggagcagttgcatcatcagcatggtatgatgttgttcagagagattttgtcgcaggcacatatgtttggacaggatttgattatctgggcgaaccgacaccgtggaatggcacaggctcaggcgcagttggctcatggccgtcaccgaaaaatagctattttggcatcgttgatacagcaggctttccgaaagatacatattatttttatcagagccagtggaatgatgatgttcatacactgcatattcttccggcatggaatgaaaatgttgttgcaaaaggctcaggcaataatgttccggttgtcgtttatacagatgcagcgaaagtgaaactgtattttacaccgaaaggctcaacagaaaaaagactgatcggcgaaaaatcatttacaaaaaaaacaacagcggcaggctatacatatcaagtctatgaaggcagcgataaagattcaacagcgcataaaaacatgtatctgacatggaatgttccgtgggcagaaggcacaatttcagcggaagcgtatgatgaaaataatcgcctgattccggaaggcagcacagaaggcaacgcatcagttacaacaacaggcaaagcagcaaaactgaaagcagatgcggatcgcaaaacaattacagcggatggcaaagatctgtcatatattgaagtcgatgtcacagatgcaaatggccatattgttccggatgcagcaaatagagtcacatttgatgttaaaggcgcaggcaaactggttggcgttgataatggctcatcaccggatcatgattcatatcaagcggataaccgcaaagcattttcaggcaaagtcctggcaattgttcagtcaacaaaagaagcaggcgaaattacagttacagcaaaagcagatggcctgcaatcaagcacagttaaaattgcaacaacagcagttccgggaacaagcacagaaaaaacagtccgcagcttttattacagccgcaactattatgtcaaaacaggcaacaaaccgattctgccgtcagatgttgaagttcgctattcagatggaacaagcgatagacaaaacgttacatgggatgcagtttcagatgatcaaattgcaaaagcaggctcattttcagttgcaggcacagttgcaggccaaaaaattagcgttcgcgtcacaatgattgatgaaattggcgcactg >SEQ ID NO: 11 nucleotide sequenceencoding BIF_1068gttgaagatgcaacaagaagcgatagcacaacacaaatgtcatcaacaccggaagttgtttattcatcagcggtcgatagcaaacaaaatcgcacaagcgattttgatgcgaactggaaatttatgctgtcagatagcgttcaagcacaagatccggcatttgatgattcagcatggcaacaagttgatctgccgcatgattatagcatcacacagaaatatagccaaagcaatgaagcagaatcagcatatcttccgggaggcacaggctggtatagaaaaagctttacaattgatagagatctggcaggcaaacgcattgcgattaattttgatggcgtctatatgaatgcaacagtctggtttaatggcgttaaactgggcacacatccgtatggctattcaccgttttcatttgatctgacaggcaatgcaaaatttggcggagaaaacacaattgtcgtcaaagttgaaaatagactgccgtcatcaagatggtattcaggcagcggcatttatagagatgttacactgacagttacagatggcgttcatgttggcaataatggcgtcgcaattaaaacaccgtcactggcaacacaaaatggcggagatgtcacaatgaacctgacaacaaaagtcgcgaatgatacagaagcagcagcgaacattacactgaaacagacagtttttccgaaaggcggaaaaacggatgcagcaattggcacagttacaacagcatcaaaatcaattgcagcaggcgcatcagcagatgttacaagcacaattacagcagcaagcccgaaactgtggtcaattaaaaacccgaacctgtatacagttagaacagaagttctgaacggaggcaaagttctggatacatatgatacagaatatggctttcgctggacaggctttgatgcaacatcaggcttttcactgaatggcgaaaaagtcaaactgaaaggcgttagcatgcatcatgatcaaggctcacttggcgcagttgcaaatagacgcgcaattgaaagacaagtcgaaatcctgcaaaaaatgggcgtcaatagcattcgcacaacacataatccggcagcaaaagcactgattgatgtctgcaatgaaaaaggcgttctggttgtcgaagaagtctttgatatgtggaaccgcagcaaaaatggcaacacggaagattatggcaaatggtttggccaagcaattgcaggcgataatgcagttctgggaggcgataaagatgaaacatgggcgaaatttgatcttacatcaacaattaaccgcgatagaaatgcaccgtcagttattatgtggtcactgggcaatgaaatgatggaaggcatttcaggctcagtttcaggctttccggcaacatcagcaaaactggttgcatggacaaaagcagcagattcaacaagaccgatgacatatggcgataacaaaattaaagcgaactggaacgaatcaaatacaatgggcgataatctgacagcaaatggcggagttgttggcacaaattattcagatggcgcaaactatgataaaattcgtacaacacatccgtcatgggcaatttatggctcagaaacagcatcagcgattaatagccgtggcatttataatagaacaacaggcggagcacaatcatcagataaacagctgacaagctatgataattcagcagttggctggggagcagttgcatcatcagcatggtatgatgttgttcagagagattttgtcgcaggcacatatgtttggacaggatttgattatctgggcgaaccgacaccgtggaatggcacaggctcaggcgcagttggctcatggccgtcaccgaaaaatagctattttggcatcgttgatacagcaggctttccgaaagatacatattatttttatcagagccagtggaatgatgatgttcatacactgcatattcttccggcatggaatgaaaatgttgttgcaaaaggctcaggcaataatgttccggttgtcgtttatacagatgcagcgaaagtgaaactgtattttacaccgaaaggctcaacagaaaaaagactgatcggcgaaaaatcatttacaaaaaaaacaacagcggcaggctatacatatcaagtctatgaaggcagcgataaagattcaacagcgcataaaaacatgtatctgacatggaatgttccgtgggcagaaggcacaatttcagcggaagcgtatgatgaaaataatcgcctgattccggaaggcagcacagaaggcaacgcatcagttacaacaacaggcaaagcagcaaaactgaaagcagatgcggatcgcaaaacaattacagcggatggcaaagatctgtcatatattgaagtcgatgtcacagatgcaaatggccatattgttccggatgcagcaaatagagtcacatttgatgttaaaggcgcaggcaaactggttggcgttgataatggctcatcaccggatcatgattcatatcaagcggataaccgcaaagcattttcaggcaaagtcctggcaattgttcagtcaacaaaagaagcaggcgaaattacagttacagcaaaagcagatggcctgcaatcaagcacagttaaaattgcaacaacagcagttccgggaacaagcacagaaaaaacagtccgcagcttttattacagccgcaactattatgtcaaaacaggcaacaaaccgattctgccgtcagatgttgaagttcgctattcagatggaacaagcgatagacaaaacgttacatgggatgcagtttcagatgatcaaattgcaaaagcaggctcattttcagttgcaggcacagttgcaggccaaaaaattagcgttcgcgtcacaatgattgatgaaattggcgcactgctgaattattcagcaagcacaccggttggcacaccggcagttcttccgggatcaagaccggcagtcctgccggatggcacagtcacatcagcaaattttgcagtccattggacaaaaccggcagatacagtctataatacagcaggcacagtcaaagtaccgggaacagcaacagtttttggcaaagaatttaaagtcacagcgacaattagagttcaa >SEQID NO: 12 nucleotide sequence encoding BIF_1172gttgaagatgcaacaagaagcgatagcacaacacaaatgtcatcaacaccggaagttgtttattcatcagcggtcgatagcaaacaaaatcgcacaagcgattttgatgcgaactggaaatttatgctgtcagatagcgttcaagcacaagatccggcatttgatgattcagcatggcaacaagttgatctgccgcatgattatagcatcacacagaaatatagccaaagcaatgaagcagaatcagcatatcttccgggaggcacaggctggtatagaaaaagctttacaattgatagagatctggcaggcaaacgcattgcgattaattttgatggcgtctatatgaatgcaacagtctggtttaatggcgttaaactgggcacacatccgtatggctattcaccgttttcatttgatctgacaggcaatgcaaaatttggcggagaaaacacaattgtcgtcaaagttgaaaatagactgccgtcatcaagatggtattcaggcagcggcatttatagagatgttacactgacagttacagatggcgttcatgttggcaataatggcgtcgcaattaaaacaccgtcactggcaacacaaaatggcggagatgtcacaatgaacctgacaacaaaagtcgcgaatgatacagaagcagcagcgaacattacactgaaacagacagtttttccgaaaggcggaaaaacggatgcagcaattggcacagttacaacagcatcaaaatcaattgcagcaggcgcatcagcagatgttacaagcacaattacagcagcaagcccgaaactgtggtcaattaaaaacccgaacctgtatacagttagaacagaagttctgaacggaggcaaagttctggatacatatgatacagaatatggctttcgctggacaggctttgatgcaacatcaggcttttcactgaatggcgaaaaagtcaaactgaaaggcgttagcatgcatcatgatcaaggctcacttggcgcagttgcaaatagacgcgcaattgaaagacaagtcgaaatcctgcaaaaaatgggcgtcaatagcattcgcacaacacataatccggcagcaaaagcactgattgatgtctgcaatgaaaaaggcgttctggttgtcgaagaagtctttgatatgtggaaccgcagcaaaaatggcaacacggaagattatggcaaatggtttggccaagcaattgcaggcgataatgcagttctgggaggcgataaagatgaaacatgggcgaaatttgatcttacatcaacaattaaccgcgatagaaatgcaccgtcagttattatgtggtcactgggcaatgaaatgatggaaggcatttcaggctcagtttcaggctttccggcaacatcagcaaaactggttgcatggacaaaagcagcagattcaacaagaccgatgacatatggcgataacaaaattaaagcgaactggaacgaatcaaatacaatgggcgataatctgacagcaaatggcggagttgttggcacaaattattcagatggcgcaaactatgataaaattcgtacaacacatccgtcatgggcaatttatggctcagaaacagcatcagcgattaatagccgtggcatttataatagaacaacaggcggagcacaatcatcagataaacagctgacaagctatgataattcagcagttggctggggagcagttgcatcatcagcatggtatgatgttgttcagagagattttgtcgcaggcacatatgtttggacaggatttgattatctgggcgaaccgacaccgtggaatggcacaggctcaggcgcagttggctcatggccgtcaccgaaaaatagctattttggcatcgttgatacagcaggctttccgaaagatacatattatttttatcagagccagtggaatgatgatgttcatacactgcatattcttccggcatggaatgaaaatgttgttgcaaaaggctcaggcaataatgttccggttgtcgtttatacagatgcagcgaaagtgaaactgtattttacaccgaaaggctcaacagaaaaaagactgatcggcgaaaaatcatttacaaaaaaaacaacagcggcaggctatacatatcaagtctatgaaggcagcgataaagattcaacagcgcataaaaacatgtatctgacatggaatgttccgtgggcagaaggcacaatttcagcggaagcgtatgatgaaaataatcgcctgattccggaaggcagcacagaaggcaacgcatcagttacaacaacaggcaaagcagcaaaactgaaagcagatgcggatcgcaaaacaattacagcggatggcaaagatctgtcatatattgaagtcgatgtcacagatgcaaatggccatattgttccggatgcagcaaatagagtcacatttgatgttaaaggcgcaggcaaactggttggcgttgataatggctcatcaccggatcatgattcatatcaagcggataaccgcaaagcattttcaggcaaagtcctggcaattgttcagtcaacaaaagaagcaggcgaaattacagttacagcaaaagcagatggcctgcaatcaagcacagttaaaattgcaacaacagcagttccgggaacaagcacagaaaaaacagtccgcagcttttattacagccgcaactattatgtcaaaacaggcaacaaaccgattctgccgtcagatgttgaagttcgctattcagatggaacaagcgatagacaaaacgttacatgggatgcagtttcagatgatcaaattgcaaaagcaggctcattttcagttgcaggcacagttgcaggccaaaaaattagcgttcgcgtcacaatgattgatgaaattggcgcactgctgaattattcagcaagcacaccggttggcacaccggcagttcttccgggatcaagaccggcagtcctgccggatggcacagtcacatcagcaaattttgcagtccattggacaaaaccggcagatacagtctataatacagcaggcacagtcaaagtaccgggaacagcaacagtttttggcaaagaatttaaagtcacagcgacaattagagttcaaagaagccaagttacaattggctcatcagtttcaggaaatgcactgagactgacacaaaatattccggcagataaacaatcagatacactggatgcgattaaagatggctcaacaacagttgatgcaaatacaggcggaggcgcaaatccgtcagcatggacaaattgggcatattcaaaagcaggccataacacagcggaaattacatttgaatatgcgacagaacaacaactgggccagatcgtcatgtatttttttcgcgatagcaatgcagttagatttccggatgctggcaaaacaaaaattcagatc >SEQ ID NO: 13 nucleotidesequence encoding BIF_1241gttgaagatgcaacaagaagcgatagcacaacacaaatgtcatcaacaccggaagttgtttattcatcagcggtcgatagcaaacaaaatcgcacaagcgattttgatgcgaactggaaatttatgctgtcagatagcgttcaagcacaagatccggcatttgatgattcagcatggcaacaagttgatctgccgcatgattatagcatcacacagaaatatagccaaagcaatgaagcagaatcagcatatcttccgggaggcacaggctggtatagaaaaagctttacaattgatagagatctggcaggcaaacgcattgcgattaattttgatggcgtctatatgaatgcaacagtctggtttaatggcgttaaactgggcacacatccgtatggctattcaccgttttcatttgatctgacaggcaatgcaaaatttggcggagaaaacacaattgtcgtcaaagttgaaaatagactgccgtcatcaagatggtattcaggcagcggcatttatagagatgttacactgacagttacagatggcgttcatgttggcaataatggcgtcgcaattaaaacaccgtcactggcaacacaaaatggcggagatgtcacaatgaacctgacaacaaaagtcgcgaatgatacagaagcagcagcgaacattacactgaaacagacagtttttccgaaaggcggaaaaacggatgcagcaattggcacagttacaacagcatcaaaatcaattgcagcaggcgcatcagcagatgttacaagcacaattacagcagcaagcccgaaactgtggtcaattaaaaacccgaacctgtatacagttagaacagaagttctgaacggaggcaaagttctggatacatatgatacagaatatggctttcgctggacaggctttgatgcaacatcaggcttttcactgaatggcgaaaaagtcaaactgaaaggcgttagcatgcatcatgatcaaggctcacttggcgcagttgcaaatagacgcgcaattgaaagacaagtcgaaatcctgcaaaaaatgggcgtcaatagcattcgcacaacacataatccggcagcaaaagcactgattgatgtctgcaatgaaaaaggcgttctggttgtcgaagaagtctttgatatgtggaaccgcagcaaaaatggcaacacggaagattatggcaaatggtttggccaagcaattgcaggcgataatgcagttctgggaggcgataaagatgaaacatgggcgaaatttgatcttacatcaacaattaaccgcgatagaaatgcaccgtcagttattatgtggtcactgggcaatgaaatgatggaaggcatttcaggctcagtttcaggctttccggcaacatcagcaaaactggttgcatggacaaaagcagcagattcaacaagaccgatgacatatggcgataacaaaattaaagcgaactggaacgaatcaaatacaatgggcgataatctgacagcaaatggcggagttgttggcacaaattattcagatggcgcaaactatgataaaattcgtacaacacatccgtcatgggcaatttatggctcagaaacagcatcagcgattaatagccgtggcatttataatagaacaacaggcggagcacaatcatcagataaacagctgacaagctatgataattcagcagttggctggggagcagttgcatcatcagcatggtatgatgttgttcagagagattttgtcgcaggcacatatgtttggacaggatttgattatctgggcgaaccgacaccgtggaatggcacaggctcaggcgcagttggctcatggccgtcaccgaaaaatagctattttggcatcgttgatacagcaggctttccgaaagatacatattatttttatcagagccagtggaatgatgatgttcatacactgcatattcttccggcatggaatgaaaatgttgttgcaaaaggctcaggcaataatgttccggttgtcgtttatacagatgcagcgaaagtgaaactgtattttacaccgaaaggctcaacagaaaaaagactgatcggcgaaaaatcatttacaaaaaaaacaacagcggcaggctatacatatcaagtctatgaaggcagcgataaagattcaacagcgcataaaaacatgtatctgacatggaatgttccgtgggcagaaggcacaatttcagcggaagcgtatgatgaaaataatcgcctgattccggaaggcagcacagaaggcaacgcatcagttacaacaacaggcaaagcagcaaaactgaaagcagatgcggatcgcaaaacaattacagcggatggcaaagatctgtcatatattgaagtcgatgtcacagatgcaaatggccatattgttccggatgcagcaaatagagtcacatttgatgttaaaggcgcaggcaaactggttggcgttgataatggctcatcaccggatcatgattcatatcaagcggataaccgcaaagcattttcaggcaaagtcctggcaattgttcagtcaacaaaagaagcaggcgaaattacagttacagcaaaagcagatggcctgcaatcaagcacagttaaaattgcaacaacagcagttccgggaacaagcacagaaaaaacagtccgcagcttttattacagccgcaactattatgtcaaaacaggcaacaaaccgattctgccgtcagatgttgaagttcgctattcagatggaacaagcgatagacaaaacgttacatgggatgcagtttcagatgatcaaattgcaaaagcaggctcattttcagttgcaggcacagttgcaggccaaaaaattagcgttcgcgtcacaatgattgatgaaattggcgcactgctgaattattcagcaagcacaccggttggcacaccggcagttcttccgggatcaagaccggcagtcctgccggatggcacagtcacatcagcaaattttgcagtccattggacaaaaccggcagatacagtctataatacagcaggcacagtcaaagtaccgggaacagcaacagtttttggcaaagaatttaaagtcacagcgacaattagagttcaaagaagccaagttacaattggctcatcagtttcaggaaatgcactgagactgacacaaaatattccggcagataaacaatcagatacactggatgcgattaaagatggctcaacaacagttgatgcaaatacaggcggaggcgcaaatccgtcagcatggacaaattgggcatattcaaaagcaggccataacacagcggaaattacatttgaatatgcgacagaacaacaactgggccagatcgtcatgtatttttttcgcgatagcaatgcagttagatttccggatgctggcaaaacaaaaattcagatcagcgcagatggcaaaaattggacagatctggcagcaacagaaacaattgcagcgcaagaatcaagcgatagagtcaaaccgtatacatatgattttgcaccggttggcgcaacatttgttaaagtgacagtcacaaacgcagatacaacaacaccgtcaggcgttgtttgcgcaggcctgacagaaattgaactgaaaacagcgaca >SEQID NO: 14 nucleotide sequence encoding BIF_1326gttgaagatgcaacaagaagcgatagcacaacacaaatgtcatcaacaccggaagttgtttattcatcagcggtcgatagcaaacaaaatcgcacaagcgattttgatgcgaactggaaatttatgctgtcagatagcgttcaagcacaagatccggcatttgatgattcagcatggcaacaagttgatctgccgcatgattatagcatcacacagaaatatagccaaagcaatgaagcagaatcagcatatcttccgggaggcacaggctggtatagaaaaagctttacaattgatagagatctggcaggcaaacgcattgcgattaattttgatggcgtctatatgaatgcaacagtctggtttaatggcgttaaactgggcacacatccgtatggctattcaccgttttcatttgatctgacaggcaatgcaaaatttggcggagaaaacacaattgtcgtcaaagttgaaaatagactgccgtcatcaagatggtattcaggcagcggcatttatagagatgttacactgacagttacagatggcgttcatgttggcaataatggcgtcgcaattaaaacaccgtcactggcaacacaaaatggcggagatgtcacaatgaacctgacaacaaaagtcgcgaatgatacagaagcagcagcgaacattacactgaaacagacagtttttccgaaaggcggaaaaacggatgcagcaattggcacagttacaacagcatcaaaatcaattgcagcaggcgcatcagcagatgttacaagcacaattacagcagcaagcccgaaactgtggtcaattaaaaacccgaacctgtatacagttagaacagaagttctgaacggaggcaaagttctggatacatatgatacagaatatggctttcgctggacaggctttgatgcaacatcaggcttttcactgaatggcgaaaaagtcaaactgaaaggcgttagcatgcatcatgatcaaggctcacttggcgcagttgcaaatagacgcgcaattgaaagacaagtcgaaatcctgcaaaaaatgggcgtcaatagcattcgcacaacacataatccggcagcaaaagcactgattgatgtctgcaatgaaaaaggcgttctggttgtcgaagaagtctttgatatgtggaaccgcagcaaaaatggcaacacggaagattatggcaaatggtttggccaagcaattgcaggcgataatgcagttctgggaggcgataaagatgaaacatgggcgaaatttgatcttacatcaacaattaaccgcgatagaaatgcaccgtcagttattatgtggtcactgggcaatgaaatgatggaaggcatttcaggctcagtttcaggctttccggcaacatcagcaaaactggttgcatggacaaaagcagcagattcaacaagaccgatgacatatggcgataacaaaattaaagcgaactggaacgaatcaaatacaatgggcgataatctgacagcaaatggcggagttgttggcacaaattattcagatggcgcaaactatgataaaattcgtacaacacatccgtcatgggcaatttatggctcagaaacagcatcagcgattaatagccgtggcatttataatagaacaacaggcggagcacaatcatcagataaacagctgacaagctatgataattcagcagttggctggggagcagttgcatcatcagcatggtatgatgttgttcagagagattttgtcgcaggcacatatgtttggacaggatttgattatctgggcgaaccgacaccgtggaatggcacaggctcaggcgcagttggctcatggccgtcaccgaaaaatagctattttggcatcgttgatacagcaggctttccgaaagatacatattatttttatcagagccagtggaatgatgatgttcatacactgcatattcttccggcatggaatgaaaatgttgttgcaaaaggctcaggcaataatgttccggttgtcgtttatacagatgcagcgaaagtgaaactgtattttacaccgaaaggctcaacagaaaaaagactgatcggcgaaaaatcatttacaaaaaaaacaacagcggcaggctatacatatcaagtctatgaaggcagcgataaagattcaacagcgcataaaaacatgtatctgacatggaatgttccgtgggcagaaggcacaatttcagcggaagcgtatgatgaaaataatcgcctgattccggaaggcagcacagaaggcaacgcatcagttacaacaacaggcaaagcagcaaaactgaaagcagatgcggatcgcaaaacaattacagcggatggcaaagatctgtcatatattgaagtcgatgtcacagatgcaaatggccatattgttccggatgcagcaaatagagtcacatttgatgttaaaggcgcaggcaaactggttggcgttgataatggctcatcaccggatcatgattcatatcaagcggataaccgcaaagcattttcaggcaaagtcctggcaattgttcagtcaacaaaagaagcaggcgaaattacagttacagcaaaagcagatggcctgcaatcaagcacagttaaaattgcaacaacagcagttccgggaacaagcacagaaaaaacagtccgcagcttttattacagccgcaactattatgtcaaaacaggcaacaaaccgattctgccgtcagatgttgaagttcgctattcagatggaacaagcgatagacaaaacgttacatgggatgcagtttcagatgatcaaattgcaaaagcaggctcattttcagttgcaggcacagttgcaggccaaaaaattagcgttcgcgtcacaatgattgatgaaattggcgcactgctgaattattcagcaagcacaccggttggcacaccggcagttcttccgggatcaagaccggcagtcctgccggatggcacagtcacatcagcaaattttgcagtccattggacaaaaccggcagatacagtctataatacagcaggcacagtcaaagtaccgggaacagcaacagtttttggcaaagaatttaaagtcacagcgacaattagagttcaaagaagccaagttacaattggctcatcagtttcaggaaatgcactgagactgacacaaaatattccggcagataaacaatcagatacactggatgcgattaaagatggctcaacaacagttgatgcaaatacaggcggaggcgcaaatccgtcagcatggacaaattgggcatattcaaaagcaggccataacacagcggaaattacatttgaatatgcgacagaacaacaactgggccagatcgtcatgtatttttttcgcgatagcaatgcagttagatttccggatgctggcaaaacaaaaattcagatcagcgcagatggcaaaaattggacagatctggcagcaacagaaacaattgcagcgcaagaatcaagcgatagagtcaaaccgtatacatatgattttgcaccggttggcgcaacatttgttaaagtgacagtcacaaacgcagatacaacaacaccgtcaggcgttgtttgcgcaggcctgacagaaattgaactgaaaacagcgacaagcaaatttgtcacaaatacatcagcagcactgtcatcacttacagtcaatggcacaaaagtttcagattcagttctggcagcaggctcatataacacaccggcaattatcgcagatgttaaagcggaaggcgaaggcaatgcaagcgttacagtccttccggcacatgataatgttattcgcgtcattacagaaagcgaagatcatgtcacacgcaaaacatttacaatcaacctgggcacagaacaagaattt >SEQID NO: 15 forward primer for generation of BIF variantsGGGGTAACTAGTGGAAGATGCAACAAGAAG >SEQ ID NO: 16 reverse primer for BIF917GCGCTTAATTAATTATGTTTTTTCTGTGCTTGTTC >SEQ ID NO: 17 reverse primer forBIF995 GCGCTTAATTAATTACAGTGCGCCAATTTCATCAATCA >SEQ ID NO: 18 reverseprimer for BIF1068 GCGCTTAATTAATTATTGAACTCTAATTGTCGCTG >SEQ ID NO: 19reverse primer for BIF1241 GCGCTTAATTAATTATGTCGCTGTTTTCAGTTCAAT >SEQ IDNO: 20 reverse primer for BIF1326GCGCTTAATTAATTAAAATTCTTGTTCTGTGCCCA >SEQ ID NO: 21 reverse primer forBIF1478 GCGCTTAATTAATTATCTCAGTCTAATTTCGCTTGCGC >SEQ ID NO: 22Bifidobacterium bifidum BIF1750vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwkfmlsdsvqaqdpafddsawqqvdlphdysitqkysqsneaesaylpggtgwyrksftidrdlagkriainfdgvymnatvwfngvklgthpygyspfsfdltgnakfggentivvkvenrlpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslatqnggdvtmnlttkvandteaaanitlkqtvfpkggktdaaigtvttasksiaagasadvtstitaaspklwsiknpnlytvrtevlnggkvldtydteygfrwtgfdatsgfslngekvklkgvsmhhdqgslgavanrraierqveilqkmgvnsirtthnpaakalidvcnekgvlvveevfdmwnrskngntedygkwfgqaiagdnavlggdkdetwakfdltstinrdrnapsvimwslgnemmegisgsvsgfpatsaklvawtkaadstrpmtygdnkikanwnesntmgdnltanggvvgtnysdganydkirtthpswaiygsetasainsrgiynrttggaqssdkqltsydnsavgwgavassawydvvqrdfvagtyvwtgfdylgeptpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqsqwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvklyftpkgstekrligeksftkkttaagytyqvyegsdkdstahknmyltwnvpwaegtisaeaydennrlipegstegnasvtttgkaaklkadadrktitadgkdlsyievdvtdanghivpdaanrvtfdvkgagklvgvdngsspdhdsyqadnrkafsgkvlaivqstkeageitvtakadglqsstvkiattavpgtstektvrsfyysrnyyvktgnkpilpsdvevrysdgtsdrqnvtwdavsddqiakagsfsvagtvagqkisvrvtmideigallnysastpvgtpavlpgsrpavlpdgtvtsanfavhwtkpadtvyntagtvkvpgtatvfgkefkvtatirvqrsqvtigssvsgnalrltqnipadkqsdtldaikdgsttvdantggganpsawtnwayskaghntaeitfeyateqqlgqivmyffrdsnavrfpdagktkiqisadgknwtdlaatetiaaqessdrvkpytydfapvgatfvkvtvtnadtttpsgvvcaglteielktatskfvtntsaalssltvngtkvsdsvlaagsyntpaiiadvkaegegnasvtvlpahdnvirvitesedhvtrktftinlgteqefpadsderdypaadmtvtvgseqtsgtategpkkfavdgntstywhsnwtpttvndlwiafelqkptkldalrylprpagskngsvteykvqvsddgtnwtdagsgtwttdygwklaefnqpvttkhvrlkavhtyadsgndkfmsaseirlrkavdttdisgatvtvpakltvdrvdadhpatfatkdvtvtlgdatlrygvdylldyagntavgkatvtvrgidkysgtvaktftielknapapeptltsvsvktkpskltyvvgdafdpaglvlqhdrqadrppqplvgeqadergltcgtrcdrveqlrkhenreahrtgldhlefvgaadgavgeqatfkvhvhadqgdgrhddaderdidphvpvdhavgelaraachhviglrvdthrlkasgfqipaddmaeidritgfhrferhvg >SEQID NO: 23 The signal sequence of extracellular lactase fromBifidobacterium bifidum DSM20215 Vrskklwisllfalaliftmafgstssaqa

1. A polypeptide having transgalactosylating activity selected from thegroup consisting of: a. a polypeptide comprising an amino acid sequencehaving at least 90% sequence identity with SEQ ID NO: 1, wherein saidpolypeptide consists of at most 980 amino acid residues, b. apolypeptide comprising an amino acid sequence having at least 97%sequence identity with SEQ ID NO: 2, wherein said polypeptide consistsof at most 975 amino acid residues, c. a polypeptide comprising an aminoacid sequence having at least 96.5% sequence identity with SEQ ID NO: 3,wherein said polypeptide consists of at most 1300 amino acid residues,d. a polypeptide encoded by a polynucleotide that hybridizes under atleast low stringency conditions with i) the nucleic acid sequencecomprised in SEQ ID NO: 9, 10, 11, 12 or 13 encoding the polypeptide ofSEQ ID NO: 1, 2, 3, 4 or 5; or ii) the complementary strand of i), e. apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 70% identity to the nucleotide sequence encoding for thepolypeptide of SEQ ID NO: 1, 2, 3, 4 or 5 or the nucleotide sequencecomprised in SEQ ID NO: 9, 10, 11, 12 or 13 encoding a maturepolypeptide, and f. a polypeptide comprising a deletion, insertionand/or conservative substitution of one or more amino acid residues ofSEQ ID NO: 1, 2, 3, 4 or
 5. 2. The polypeptide according to claim 1comprising an amino acid sequence having at least 90% sequence identitywith SEQ ID NO: 1, wherein said polypeptide consists of at most 980amino acid residues.
 3. The polypeptide according to claim 1 comprisingan amino acid sequence having at least 97% sequence identity with SEQ IDNO: 2, wherein said polypeptide consists of at most 975 amino acidresidues.
 4. The polypeptide according to claim 1 comprising an aminoacid sequence having at least 96.5% sequence identity with SEQ ID NO: 3,wherein said polypeptide consists of at most 1300 amino acid residues.5. The polypeptide according to claim 1, wherein said polypeptideconsists of the amino acid sequence selected from SEQ ID NO: 1, SEQ IDNO:2, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO:
 4. 6. The polypeptideaccording to claim 1, wherein said polypeptide consists of the aminoacid sequence selected from SEQ ID NO:1, or SEQ ID NO:
 2. 7. Thepolypeptide according to claim 1, wherein said polypeptide has a ratioof transgalactosylation activity above 100%.
 8. A nucleic acid capableof encoding a polypeptide according to claim
 1. 9. An expression vectorcomprising a nucleic acid according to claim
 8. 10. A cell capable ofexpressing a polypeptide according to claim
 1. 11. A method ofexpressing a polypeptide, the method comprising obtaining a cellaccording claim 10 and expressing the polypeptide from the cell, andoptionally purifying the polypeptide.
 12. A composition comprising apolypeptide as defined in claim 1, preferably a food composition, morepreferably a dairy product.
 13. A method for producing a food product bytreating a substrate comprising lactose with a polypeptide as defined inclaim
 1. 14. A method for producing a dairy product by treating amilk-based substrate comprising lactose with a polypeptide as defined inclaim
 1. 15. A process for producing galacto-oligosaccharides,comprising contacting a polypeptide of claim 1 with a milk-basedsolution comprising lactose.
 16. A process for producinggalacto-oligosaccharides, comprising contacting a purified polypeptideof claim 11 with a milk-based solution comprising lactose.